Environ. Sci. Technol. 1907, 21, 149-155
Solubility Properties in Polymers and Biological Media. 8. An Analysis of the Factors That Influence Toxicities of Organic Nonelectrolytes to the Golden Orfe Fish (Leuciscus idus melanotus) Mortimer J. Kamlet” and Ruth M. Doherty Naval Surface Weapons Center, White Oak Laboratory, Silver Spring, Maryland 20910
Robert ,W. Taft Department of Chemistry, University of California, Irvine, California 927 17
Mlchael H. Abraham Department of Chemistry, University of Surrey, Guildford, Surrey GU2 5XH, United Kingdom
Gilman D. Veith Environmental Research Laboratory-Duluth,
U.S. Environmental Protection Agency, Duluth, Minnesota 55804
Donald J. Abraham Department of Medicinal Chemistry, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 1526 1
Toxicities of organic nonelectrolytes to the golden orfe fish are well correlated ( n = 32, r = 0.983, SD = 0.19) by log LC50(mmol/L) = 3.19 - 3.29P/100 - 1 . 1 4 ~ ”+ 4.600 - 1 . 5 2 ~ ~ where is the solute molar volume and T * , 6, and a, are the solvatochromic parameters that measure dipolarity/ polarizability, hydrogen-bond acceptor basicity, and hydrogen-bond donor acidity of the solute (toxicant). I t is suggested that carboxylic acid esters are more toxic than predicted by the above equation because of in vivo hydrolysis and that alkanes, alkylbenzenes, and chlorobenzenes are less toxic than predicted because of sequestration in hydrophobic pockets in fish blood hemoglobin.
Introduction The present series of papers has as its purpose to demonstrate that many disparate and seemingly unrelated physicochemical, biochemical, pharmacological, and toxicological properties that depend on solute-solvent interactions can be correlated, rationalized, and predicted in terms of a single generalized linear solvation energy relationship of simple and conceptually explicit form. In an earlier paper of this series ( I ) , we reported the application of this methodology to the correlation of inhibition of bioluminescence in Photobacterium phosphoreum (the Microtox test). In this paper we carry out a similar analysis of factors influencing toxicities of nonelectrolytes to the golden orfe fish (Leuciscus idus melanotus). We present a simple equation for correlation and prediction of toxicities of compounds that act by a general nonreactive toxicity mechanism, and we suggest reasons why certain compounds are more toxic and others are less toxic than predicted by that equation. We also discuss unexpected parallelisms between toxicities of some hydrophobic nonelectrolytes to the golden orfe fish and solubilities of those same nonelectrolytes in human blood. These toxicological studies are an outgrowth of our earlier findings that solubility properties, SP, of nonelectrolyte (2-6) and electrolyte (7) solutes in a variety of media can be expressed by linear combinations of terms that estimate the endoergic requirements of the cavityforming process and the exoergic effects of solute-solvent dipolar and hydrogen-bonding interactions. The cavity term measures the free energy expenditure necessary to 0013-936X/87/0921-0149$01.50/0
separate the solvent molecules and provide a suitably sized cavity for the solute. The solute property that influences this term is its molar volume, taken here as its molecular weight divided by its liquid density at 20 OC. The com~plementary solvent property is (6H2)1, the square of the Hildebrand solubility parameter (8, 9) (we use the convention that subscript 1 applies to the solvent and subscript 2 to the solute). A dipolarity/polarizability term, which measures the exoergic effects of solute-solvent dipolar interactions, depends on T * and ~ T * ~T* , being the solvatochromic parameter (9-12) that describes the ability of a compound to stabilize a neighboring charge or dipole by virtue of nonspecific dielectric interactions. For hydrogen bond acceptor (HBA) solutes in hydrogen bond donor (HBD) solutes like water, the exoergic effects of solvent to solute hydrogen bonding are measured by terms in al and (&J2, a and 0 being the solvatochromic parameters that scale HBD acidity and HBA basicity and the subscript m indicating that for amphihydrogen-bonding compounds the “monomer” value (i.e., the value applicable to the nonself-associated solute) ( 3 , 5 , 1 3 )is used. For HBD solutes in HBA solvents, hydrogen-bondinginteractions may also lead to a term in (a,), and pl. For non-self-associating compounds, p = p,. Accordingly, the generalized equations describing most linear solvation energy relationships take the form of eq 1,where any one or combination of terms may drop out SP = SPO + A(a2)1v* + B T * ~ T+* ~ca,(Prn), + oPl(arn)z (1) if not applicable to the property studied. When dealing with the properties of a single solute or set of reactants in a series of solvents, the factors relating to the solutes are subsumed into the constants in eq 1,and the resulting eq 2 relates specifically to the solvent parameters. ConSP = SPo + h(6H2)1/100 + ST*^ + a ( ~ + 1 bpi (2) versely, when dealing with properties of multiple solutes in single solvents or with distributions between solvents (as in the present case), the equation relates specifically to the solute parameters, eq 3. We use (6H2)1/100and SP = SPo+ mvz/lOO + ST*, + a(a& + b(&), (3)
P, J 100 in order that the scale measuring the cavity term should be similar to those for the other terms, which makes
@ 1987 American Chemical Society
Environ. Sci. Technol., Vol. 21, No. 2, 1987
149
easier the evaluation of the contributions of the various solute-solvent interactions to the solubility property studied. Equation 2 has been used in hundreds of correlations of solvent effects on such diverse properties as positions and intensities of maxima in UV/visible, IR, ESR, and NMR spectra, reaction rate and equilibrium constants, fluorescence lifetimes, formation constants of hydrogenbonded and Lewis acid/base complexes, and free energies of solution and transfer between solvents of electrolyte and nonelectrolyte solutes (14,15). Equation 3 was arrived at more recently, and to date, its most important applications have been in the correlations of aqueous solubilities of 105 liquid aliphatic nonelectrolytes (r = 0.995, SD = 0.14) (2, 3) and octanol/water partition coefficients of 102 aliphatic and aromatic non-HB, HBA, and weak HBD solutes ( r = 0.989, SD = 0.18) (4, 5 ) . We have also recently reported (16) that solubilities in human blood at 37 “C, or, more precisely, values of S,K(blood/gas), where K(blood/gas), is the Ostwald coefficient on blood and S, is the solute concentration in its owp saturated vapor at 37 OC (S, = Patm/25.4),are well correlated by eq 4 ( n = 27, r = 0.987, SD = 0.14). The data set logS(b1ood) log [S&blood/gas)] = 1.35 - 3 . 0 5 ~ ~ / 1 0-00.22 i ~ * z 3.58(pm)2 (4)
+
leading to eq 4 included non-hydrogen-bonding,HBA, and weak HBD solutes, but eq 4 specifically did not apply to a number of alkanes, alkylbenzenes, and chlorobenzenes, which were more soluble in blood than predicted (a fact that will be shown to be highly germane to this study). Numerous workers have correlated such chemical properties as solubilities in water (S,) and octanol/water partition coefficients (KO,) with pharmacological and toxicological effectiveness in a field of biochemistry that has come to be known as quantitative structure-activity relationships (QSAR). The connection of toxicity to S, is that increasing aqueous solubility decreases absorption and increases excretion in mammals and aquatic organisms, thus lessening the tendency of the solute to build up to toxic concentrations. Increasing lipophilicity (more correctly, hydrophobicity), which is considered to covary with KO,,leads to greater concentration in and easier passage through membranes and greater distribution from blood into more lipid-like areas of the organism. Before proceeding with the present correlations, it is necessary to emphasize that two types of toxicological mechanisms will be discussed here. The correlations presented below do not apply to toxicants that act by reactive toxicological mechanisms, Le., those that involve specific chemical combination, such as Schiff-base formation between aldehyde toxicants and enzyme amine groups, eq 5, or Michael additions of enzyme sulfhydryl enzyme-NHz O=CH-toxicant enzyme-N=CH-toxicant + HzO (5)
+
enzyme-SH
-+
+ CHz=CHCOCH3
-
enzyme-SCH2CH2COCH3 (6)
groups with conjugated double bonds, as with acrolein or methyl vinyl ketone, eq 6 (17) [or unsaturated alcohols, which can be oxidized in vivo to conjugated carbonyl compounds, according to Lipnick’s “proelectrophile” mechanism (IS)]. They refer rather to nonreactive toxicological behavior, where toxic effects are shown by any organic nonelectrolyte if present in sufficient concentration and wherein pharmacokineticfactors like partitioning and transport are rate controlling. As with most nonreactive toxicological QSARs 150
Envlron. Sci. Technol., Vol. 21, No. 2, 1987
of simple molecules (19), the mechanism is usually considered to involve inhibition of physiological activity by absorption in nerve membranes, hindering electrolyte transport across those membranes, and leading to lethargy, anesthesia, narcosis, and death.
i
Results and Discussion The golden orfe toxicity data used in this paper are those of Juhnke and Ludemann (20), who reported 48-h static LCo,LC50, and LClWvalues for 200 chemical compounds. Parallel determinations for many of these toxicants were carried out by Juhnke at the Landesanstalt fur Wasser und Abfall NW (LWANW) and by Ludemann at the Institut fur Wasser, Boden-, und Lufthygiene des Bundesgezuntheitamtes Berlin (WaBoLu Berlin). Agreement between the two sets of data was only fair, and because the LWANW data set was more comprehensive, we have centered our analysis on the LC50results of Juhnke, with occasional parallel correlations of the Ludemann data to confirm the trends. For reasons that will become evident in subsequent discussions, we have peremptorily excluded certain classes of compounds from the general toxicity correlation. These include (a) amines and carboxylic acids, which are strong proton-transfer acids and bases (where the present correlations involve proton sharing); (b) aldehydes and Michael reaction addends, whose toxicities are due to specific chemical reactions, as discussed above; (c) carboxylic acid esters, which are subject to hydrolysis in vivo; and (d) alkanes, alkylbenzenes, and chlorobenzenes, some of which have been shown to be less toxic than predicted from correlations with octanol/water partition coefficients (21). This left us with 32 compounds for which the solvatochromic parameters are known or can be reliably estimated. LC5,, values (in pmol/L) are assembled in Table I for 13 alcohols, 4 ethers, 4 ketones, 4 polychloroalkanes and alkenes, 2 phenols, 2 nitro compounds, 2 nitriles, and benzene. Also included in the table are values of v2/100, T * ~pZ , or (/3m)z, and (am)2 We use the same “ground rules” as were set forth in our correlations of octanol/water partition coefficients (5);i.e., an increment of 0.10 is added to v2/100 of aromatic and alicyclic compounds; we use p2 = 0.10 for chloroaliphatic solutes; 0.10 is added to PI of anisole to account for hydrogen bonding to both the oxygen and the ring, leading to pz = 0.32. The LSER/QSAR relationship for the 32 compounds that are believed to act by the general nonreactive toxicity mechanism is given for Juhnke’s data by eq 7. Ludemann log(l/LC,o) (pmol/L) = 43.19 0.22) + (3.29 f 0.15)v2/100 + (1.14 f 0.18)~*2(4.60 f 0.25)& + (1.52 f O.20)(~1,)2 (7) n = 32, r = 0.983, SD = 0.19 has reported LC,, values for 27 of the 32 compounds in Table I plus ethanol. The correlation for this data set leads to eq 8. Although the goodness of fit to each of the lOg(l/LCBo) = - (3.73 f 0.27) + (3.16 f 0.15)~~//100 + (1.65 f 0 . 2 2 ) ~-” ~ (4.03 f 0.32)& + (2.09 f 0.23)(am)2 (8) n = 28, r = 0.981, SD = 0.21
equations is excellent by any reasonable standards one might care to apply, the correspondence between the two equations (or, indeed, between the raw data) is only fair. This may reflect intrinsic imprecision of the measurements or unspecified differences in the experimental conditions. I t is noteworthy, however, that the two equations show
Table I. Data Used in Correlation of Nonreactive Toxicological Effects no. 1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
vpoo
T*
1.046 1.693 0.757 0.765 0.915 0.920 0.917 1.086 1.094 1.256 1.255 1.244 1.414 1.575 1.009d 0.897 0.989 0.968 0.787 0.911d 0.734 0.895 1.136d 0.986d 0.521 1.163d (0.989)f 1.186d 0.98gd 1.127d 1.120d 1.27gd
0.27 0.26 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.53 0.49 0.28 0.91 0.58 0.71 0.67 0.76 0.76 0.75 (0.75)f (0.75)f 0.73 0.59 1.01 0.90 (0.90)f
toxicant diethyl ether di-n-butyl ether 1-propanol 2-propanol 1-butanol 2-methyl-1-propanol 2-butanol 1-pentanol 2-methyl-2-butanol 1-hexanol 2-hexanol 3-hexanol 1-heptanol 1-octanol cyclopentanol trichloroethylene l,l,l-trichloroethane carbon tetrachloride l,2-dichloroethane tetrahydrofuran acetone 2-butanone cyclohexanone cyclopentanone acetonitrile m-cresol phenol aniso1e benzene nitrobenzene benzonitrile 2-nitrotoluene
P or P m
am
exptl
eq 7
diff"sb
0.47 0.46 0.45 0.51 0.45 0.45 0.51 0.45 0.57 0.45 0.51 0.51 0.45 0.45 0.51
0.00 0.00 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.00 0.00 0.00
1.58 -0.26 1.88 2.17 1.21 1.31 1.67 0.74 1.44 0.10 0.45 0.54 -0.45 -0.81 1.24 0.01 -0.04 -0.21 0.56 1.59 2.29 1.80 0.74 1.58 2.15 -0.75 -0.58 0.05 -0.37 -0.31 -0.02 -0.67
1.61 -0.55 1.82 2.07 1.20 1.28 1.57 0.73 1.26 0.17 0.45 0.49 -0.35 -0.98 1.26 0.10 -0.16 0.15 0.14 2.17 2.18 1.70 1.03 1.48 2.01 -0.81 -0.33 -0.07 -0.27 -0.28 0.10 -0.66
-0.03 0.29* 0.06 0.10 0.01 0.03 0.10 0.01 0.18 -0.11 0.00 0.05 -0.10 0.08 -0.02 -0.09 0.12 -0.36* 0.42** -0.48** 0.11 0.10 -0.29* 0.10 0.14 0.06 -0.25* 0.12 -0.10 -0.03 -0.12 -0.01
0.10e
0.10e 0.10e 0.10e
0.00 0.00 0.00
0.55 0.48 0.48 0.53 0.52 0.35 0.33 0.33 0.328 0.10 0.30 0.35 (0.30)f
0.00 0.00 0.00 (0.15)f 0.55 0.61 0.00 0.00 0.00 0.00 0.00
*
"Experimental minus calculated. (*) Denotes difference of more than 1 SD; (**) denotes difference of more than 2 SD of eq 7. 'LCSO is in millimoles per liter. dAn increment of 0.10 is added to v / l O O of alicyclic and aromatic compounds, as set forth earlier (5). use P = 0.10 for chloroaliphatic solutes, as set forth earlier (5).f These values are estimated from corresponding values for closely related compounds. SA value of 0.10 is added to j3 for hydrogen bonding to the ring according to the ground rules set forth earlier (5).
Table 11. Data for Compounds That Exhibit Additional Toxicological Effects eq 7
A log LC50
Carboxylic Acid Esters 0.978 0.55 0.45 1.150 0.48 0.45 1.170 0.50 0.45 1.326 0.46 0.45 1.322 0.46 0.45 1.340 0.46 0.45 1.487 0.46 0.45 0.969 0.55 0.45 1.146 0.47 0.45 1.324 0.46 0.45 1.363d 0.53 0.44 0.924 0.55 0.45
0.58 0.28 0.55 0.08 0.03 0.46 0.00 0.34 0.09 -0.17 -1.02 -0.52
1.42 0.94 0.84 0.37 0.39 0.33 -0.16 1.44 0.95 0.38 0.13 1.59
-0.84 -0.66 -0.29 -0.29 -0.36 0.13 0.16 -1.10 -0.86 -0.55 -1.15 -2.11
0.562 0.882 1.119
Aldehydes 0.67 0.63 0.92
0.42 0.40 0.44
0.50 0.27 -0.23
2.50 1.41 0.48
-2.00 -1.14 -0.71
acrolein acrylonitrile methyl acrylate methyl methacrylate
0.666 0.658 0.902 1.060
Michael Addends 0.65 0.75 0.53 0.50
0.42 0.35 0.45 0.45
-1.36 -0.27 -1.05 0.21
2.19 1.78 1.69 1.20
-3.55 -2.05 -2.74 -0.99
n-butylamine acetic acid butyric acid
Amines and 0.987 0.575 0.925
Carboxylic Acids 0.32 0.69 0.45 c 0.46 d
0.50 0.83 0.74
2.75 2.32 1.33
-2.25 -1.49 -0.59
toxicant
33 34 35 36 37 39 40 41 42 43 44
ethyl acetate 1-propyl acetate 2-propyl acetate 1-butyl acetate 2-methyl-1-propyl acetate 2-methyl-2-propyl acetate 1-pentyl acetate methyl propionate ethyl propionate ethyl butyrate phenyl acetate vinyl acetate
45 46 47
acetaldehyde butyraldehyde benzaldehyde
48 49 50 51 52 53 54
38
log LCSO
v/100
exptl
no.
p"
"Most ?r* and P values are estimated from corresponding values for closely related compounds. *Includes an increment of 0.10 for aromatic ring. ' w = 0.57; a, = 0.71. Use of w in place of P in correlations will be discussed in a future paper. d w = 0.56; a, = 0.60.
Environ. Sci. Technol., Vol. 21, No. 2, 1987
151
Table 111. Data for Alkanes, Alkylbenzenes, and Chlorobenzenes Whose Toxicities Are Less Than Predicted by General Toxicity Equation no.
toxicant
c/100
55 56 57 58 59 60 61 62 63 64 65 66
hexane heptane cyclohexane benzene toluene xylene ethylbenzene isopropylbenzene tert-butylbenzene 1,2-diethylbenzene chlorobenzene 1,2-dichlorobenzene
1.306 1.465 1.138O 0.989Q 1.163O 1.330" 1.324O 1.489O 1.649" 1.625" 1.118" 1.227O
T*
-0.08 -0.08
0.00 0.59 0.54 0.47 0.48 0.47 0.42 0.42 0.71 0.80
P
exptl
0.00
0.32
0.00 0.00 0.10 0.11 0.12 0.12 0.12 0.12 0.12 0.07 0.04
0.39 -0.18 -0.37 -0.11 -0.09 -0.38 -0.40 -0.34 -0.62 -0.67 -0.70
log LC50 eq 7
A
-1.02 -1.54 -0.55 -0.27 -0.75 -1.17 -1.16 -1.69 -2.16 -2.08 -0.96 -1.57
1.34 1.95 0.37 -0.10 0.64 1.08 0.78 1.29 1.32 1.42 0.29 0.87
blood soly A log S b 1.30 1.41 0.49 0.07 0.27 0.87 0.70 1.06 0.44 1.05
Includes increment of 0.10 for cyclic compounds.
similar dependences on the independent variables. Further, the signs and relative magnitudes of the terms in eq 7 and 8 are the same as had been observed in our earlier LSER/QSAR studies on Photobacterium phosphoreum ( I ) , with the leading terms influencing nonreactive toxicity again being toxicant HBA basicity and molar volume. Increasing HBA basicity, p2, which favors solubility in water (whose a1value is higher than those of the protein or lipid components of the golden orfe), again leads to lower toxicity, whereas increasing solute molar volume, which favors distribution into the golden orfe [whose ( ~ 3 value ~ ~ ) is~lower than that of water], again leads to greater toxicity. Also, since lipid and protein p1values are higher than p1of water, increasing toxicant (a,), was expected to and, indeed, does favor distribution into the golden orfe, and hence greater toxicity. Only in the case of the dipolarity/polarizability term does the relationship between solubility and toxicity break down. As with the photobacterium, increasing T*,, which leads to increasing solubility in water, also leads to greater toxicity to the organism. We have seen this effect in other LSER/QSAR studies, including toxicity to the fathead minnow, the Madison 517 fungus, and the tadpole narcosis (22), and we have suggested that it may provide a clue toward the mechanism of nonreactive toxicity or narcosis ( I ) . It is attractive to consider that the effect might arise from specific charge/dipole or dipole/dipole interactions between the more toxicants and ionic acetylcholine (A), (CH3)3N+CHzCH20COCH3
(CH~),N+CH,CH,O-
B
A
CHzOCOR
I CHOCOR 0 I I CHzOP-OCH2CH,N+(CH3)3 II
Q C
zwitterionic choline (B), and/or zwitterionic lecithin (C), which is found in all living organisms and is a significant constituent of nervous tissue and brain substance. Enhanced Toxicities of Carboxylic Acid Esters. We next direct our attention to a number of compounds that are more toxic than predicted by eq 7. Data for 25 such toxicants of five general classes are assembled in Table 11, the datum of greatest interest being the A log LCb0value, Le., the difference between the observed value and that predicted by eq 7 . A negative A value indicates a greater than predicted toxicity. We have discussed the reactive toxicological properties of the aldehydes and Michael 152
Environ. Sci. Technol., VoI. 21, No. 2, 1987
addends earlier and in connection with the Microtox test correlation ( I ) , and we have no further useful comments regarding toxicants 45-51 of Table 11. Nor have we any comments regarding n-butylamine or acetic acid, whose greater than predicted toxicities are likely consequences or their greater proton-transfer acidities or basicities than those of the other toxicants of Tables 1-111 (or, in the case of n-BuNH,, reactive metabolites such as hydroxylamines, or in vivo transamination reactions with polypeptides may play a part). The lethality data for carboxylic acid esters 33-44 are of interest because the data are consistent with a mechanism involving in vivo hydrolysis. Although we do not have explicit evidence, it is seen in Table I1 that the -A log LC50 values are large for vinyl acetate and phenyl acetate and, among the alkyl acetates, decrease in magnitude with increasing chain length and chain branching, reaching essentially nil for n-pentyl and tert-butyl acetates. This ordering is consistent with neutral or alkaline in vivo hydrolysis, and it is instructive to compare some of the A values in Table I1 with log rate constants, relative to methyl acetate, for alkaline hydrolysis in water at 25 "C [from Hammett (23)]: ester
A log LCbo
CH&OOCH=CH, CH3COOCeH5 CH3COOCZH5 CH,COOCH&HzCH3
-2.11 -1.15 -0.84 -0.66 -0.29 0.13
CH3COOCH(CH3), CH,COOC(CHJB
log k / k , 1.76 0.88
-0.22 -0.26 -0.84 -2.08
The correlation coefficient for the linear regression of A log LC50 with log k / k o is 0.975. (One should realize, of
course, that there is an intrinsic imprecision in such a correlation in that A log LCs0 has a limiting value of essentially nil, while log k/kohas no such lower limit). These data are an example where esters that hydrolyze rapidly show greatly enhanced toxicity, while esters that hydrolyze more slowly show lesser toxicity and, in the extreme, follow the nonreactive toxicity mechanism predicted by eq 7. In contrast with these data for the golden orfe, observed toxicities of ethyl acetate and ethyl propionate to P. phosphoreum (the Microtox test) agree well with the QSAR for nonreactive toxicity ( I ) , which suggests that in vivo hydrolysis is slower in P. phosphoreum than in the golden orfe. On the other hand, the A log LC50 values, relative to the nonreactive toxicological correlation equation for the fathead minnow are -1.10 for n-butyl acetate (compared to -0.29 for the golden orfe) and -0.80 for nhexyl acetate, which indicates that in vivo hydrolysis is more rapid in the minnow than in the orfe. These results
R -0.825
ALOG SB
Figure 1. Differences between observed and predicted toxicities to golden orfe plotted against corresponding differences between observed and predicted solubilities in human blood.
suggest that the carboxylic acid esters are a class of compounds whose lethality properties should not be extrapolated from species to species unless relative carboxylesterase activities for the species are known. Compounds with Less T h a n Predicted Toxicities. Of greatest interest among the compounds tested by Juhnke and Ludemann (for reasons that will be seen) are a series of alkanes, alkylbenzenes, and chlorobenzenes that are less toxic than predicted by either eq 7 or an earlier correlation of log LC50with log KO, (21). Lipnick and Dunn (21) have attributed this lesser toxicity in the case of the alkane toxicants to loss by evaporation of the more volatile solutes from water under the conditions of the static test. We feel that such an effect may contribute in part to the apparent decreased toxicities of hexane and heptane (whose reported LC50 values are higher than their solubilities in water) but not in the case of the alkyl- and chlorobenzenes, all of which have lower vapor pressures and gaslwater partition coefficients than compounds like CC14,CHC1=CCl2, and CH,CC13, which fit eq 7 quite well. Observed and calculated LC50 data for the less-toxicthan-predicted compounds are assembled in Table I11 together with solvatochromic parameters of the toxicants and values of the differences between observed and calculated toxicities (A log LCw). We have noted earlier (16) that many of these same compounds are more soluble in human blood than predicted by eq 4, and we have also included in Table I11 the A log S(b1ood) values, the enhanced (relative to eq 4) solubilities in human blood. It is seen in the table that the -A log LCmand the A log S(b1ood) terms appear to follow remarkably similar trends. Increases are observed in both series with increasing alkane chain length, number of side-chain carbons in the alkylbenzenes, and number of chlorine atoms in the chlorobenzenes. Indeed, the two sets of A values show fair linear regression. The correlation is given by eq 9 and a plot is A log LC50 = 0.09 - 1.24[A log S(b100d)l (9) n = 10, r = 0.925, SD = 0.24 shown in Figure 1. In addition, log LCw and log S(b1ood) data are available for 12 compounds that fit both eq 4 and 7 and 9 compounds that fit neither equation. Again, both the conforming and the nonconforming data points show fair linear regression with one another, the correlation being given by eq 10. log LC60 = 1.33 + 1.02[10g S(b100d)l (10) n = 21, r = 0.924, SD = 0.37 Equations 9 and 10 are not precise relationships, nor would one expect them to be in the light of the different
I
4
-3
-2 -1 0 LOG S b CALCULATED
1.0
2.0
Figure 2. Solubilities in human blood plotted against values calculated through eq 4. The filled circles are for the alkanes, alkylbenzenes, and chlorobenzenes. Dashed lines correspond to probable range of concentrations of hemoglobin hydrophobic pockets.
coefficients of T * and ~ Pz in eq 4 and 7. If, however, one is willing to follow us in the as yet unsubstantiated assumption that solvent properties of fish blood are similar to those of human blood, the parallelism in eq 10 and the near proportionality in eq 9 make some sense. If the toxicological mechanism involves distribution between blood and more lipid-like nerve membranes or between blood and a hydrophobic pocket in an enzyme (24), then greater than predicted solubility in blood would be expected to lead to slower (but not lower) concentration at the site of toxic activity and hence to less than predicted toxicity. Thus the -A log LC50 and the A log S(b1ood) terms may both be manifestations of what we had earlier referred to as an anomalous sanguiphilic effect of the higher alkanes, alkylbenzenes, and chlorobenzenes. Hydrophobic Solubility Reservoir i n Blood. We propose that the excess solubilities in blood are due to sequestering of thermodynamically less soluble solutes (of appropriate sizes and shapes) in a nondipolar solubility reservoir provided by hydrophobic pockets in the hemoglobin (25-29). Although some of the pockets are, in fact, water-filled channels, they behave effectively as vacuums since, regardless of whether the hydrophobic solute fills a vacuum or displaces water solute, the cavity term is essentially nil, so that the process is energetically highly favored. Hemoglobin is present in human blood in concentrations of (2.1-2.4) X M, and there are between four and nine pockets in the hemoglobin molecule (26))so that the concentration of the pockets in whole blood is (0.8-2.2) X lo-’ M. We postulate that these pockets provide a “solubility s i n k for lipophilic solutes, even the more soluble ones, but that the effects of this solubility sink may not become statistically evident (Le., outside the normal scatter of the data) until the calculated log solubilities are lower than -1.5. In Figure 2 we show a plot of log solubilities in human blood against values calculated through eq 3. The filled circles represent the alkanes, alkylbenzenes, and chlorobenzenes,.and the horizontal dashed lines represent the probable range of concentrations of the hemoglobin pockets in whole blood. It is seen that the filled circles are for the only compounds studied with calculated log solubilities lower than -1.5 and that the experimentally observed solubilities level off at concentrations near to those of the hydrophobic pockets. For hydrocarbons with calculated log solubilities higher than -1.5, the filled circles conform closely to the correlation equation. [An exactly Environ. Sci. Technol., Vol. 21, No. 2, 1987
153
2
% /
/
I
-2.0
-1.0
I
I
I
0
1.0
2.0
/
LOG LCWVALUES CALCULATED Figure 3. Toxicities to golden orfe plotted against values calculated through eq 7. The filled circles are for alkanes, alkylbenzenes, and chlorobenzenes.
analogous leveling off at about the same concentrations was also shown in a plot of log S(b1ood) against log octanol/water partition coefficient (16).] We take these results to mean that the sanguiphilic effect is not necessarily unique to the alkanes, alkylbenzenes, and chlorobenzenes but may apply also to any solute of appropriate size, shape, and calculated solubility. [Referees of this and the earlier paper (16) have suggested as an alternative possibility that the sanguiphiIic effect might be due to binding of hydrophobic solutes to serum albumin, as described by Helmer, Kiehs, and Hansch (30). We acknowledge that this is, indeed, a possibility but prefer the present rationale for the following reasons: (i) As is seen in Figure 2, and inferentially in Figure 3, the solubilities level off at values that are remarkably close to the concentration range of the hydrophobic pockets. (ii) Compounds with large differences in P, but similar values of A* and p, show comparable values of S(b1ood) and LCEO,i.e., little or no dependence of solubility or toxicity on solute molar volume. By way of contrast, Leo, Hansch, and Church (31) have shown that complexation with bovine serum albumin (BSA) is well correlated with log Kow( r = 0.95, SD = 0.16); log KO, depends strongly on V. Further, we shall show in a future paper that the same property correlates well with the solvatochromic parameters, according to eq 11 (where VI log (l/C)(BSA) = 3.30 + 3.43V1/100 - 0.78~"- 3.676,
+ 0 . 3 7 ~(11) ~~
n = 16, r = 0.978, SD = 0.13 is the computer-calculated intrinsic molar volume). It is seen that complexation with bovine serum albumin also depends strongly on solute size. For these reasons, we believe that complexation with serum proteins does, indeed, influence S(b1ood) but that the effect is already accounted for in eq 4. Hence, in our view, the nonconformance with eq 4 requires an alternative explanation, such as is provided here.] Explanation of the Less Than Predicted Toxicities. A plot of observed golden orfe LC50 values against values calculated by eq 7 is given in Figure 3, which is seen to resemble Figure 2 quite closely. Again, the filled circles represent the alkanes, alkylbenzenes, and chlorobenzenes, and in this instance, the dashed lines represent the range of log LCb0values which, according to eq 10, correspond to S(b1ood) values of (0.8-2.2) X M (the concentration of hydrophobic pockets). It is seen that, except in the case 154
Environ. Scl. Technol., Vol. 21, No. 2, 1987
of hexane and heptane (55 and 56), where the excess volatilities mentioned by Lipnick and Dunn (21) may obtrude, the results follow the same pattern of behavior as was seen in Figure 2, presumably for the same reasons. [Interestingly, although Juhnke's LC50values for 55 and 56 already exceed their aqueous solubilities, Ludemann's LC50 values for these compounds and cyclohexane are 10-20 times higher, which suggests that these specific results are best ignored.] Hence, again with the as yet unproven proviso that hemoglobin hydrophobic pocket concentrations in fish blood are similar to those in human blood, the lower than predicted toxicities are fully explained in terms of conformance with eq 7 for the more soluble toxicants and sequestration in the hydrophobic pockets for the less soluble ones. Only after concentrations of the less soluble toxicants in fish blood exceed the concentrations of the hemoglobin pockets do those toxicants become rapidly available at the sites of toxic activity. From this an interesting question logically follows. I f the solubility reservoir provided by the hemoglobin pockets of golden orfe blood shields the fish from the harmful effects of the less soluble toxicants, does the similar reservoir in h u m a n blood mitigate or otherwise influence the therapeutic effects of less soluble hydrophobic pharmaceuticals (e.g., by providing a natural timed-release mechanism)? We intend to explore this question further in future work. A referee has pointed out that the dissolving capacity of blood can only delay the toxic event, not lead to a lower ultimate concentration; eventually, the water, blood, and the target must reach equilibrium. We agree that if LCs0 is not influenced by transport kinetics, or by competition between transport and metabolism, the above rationale may not apply.
Acknowledgments We are most grateful to N. P. Frank and W. R. Lieb of Imperial College for useful suggestions. Registry No. 1,60-29-7; 2, 142-96-1;3,71-23-8; 4,67-63-0; 5, 71-36-3; 6,78-83-1; 7,78-92-2; 8,71-41-0; 9, 75-85-4; 10, 111-27-3; 11,626-93-7; 12,623-37-0; 13,111-70-6; 14,111-87-5; 15,96-41-3; 16,79-01-6; 17,71-55-6; 18,56-23-5; 19, 107-06-2; 20,109-99-9; 21, 67-64-1; 22, 78-93-3; 23, 108-94-1; 24, 120-92-3; 25, 75-05-8; 26, 108-39-4; 27, 108-95-2; 28, 100-66-3; 29, 71-43-2; 30,98-95-3; 31, 100-47-0;32,88-72-2; 33, 141-78-6; 34,109-60-4; 35, 108-21-4;36, 123-86-4; 37,110-19-0; 38,540-88-5; 39, 628-63-7; 40,554-12-1; 41, 105-37-3;42, 105-54-4; 43, 122-79-2;44, 108-05-4;45, 75-07-0; 46, 123-72-8;47, 100-52-7; 48, 107-02-8;49, 107-13-1;50, 96-33-3; 51, 80-62-6; 52, 109-73-9; 53, 64-19-7; 54, 107-92-6; 55, 110-54-3; 56, 142-82-5; 57,110-82-7; 58,71-43-2; 59, 108-88-3; 60,1330-20-7; 61, 100-41-4; 62, 98-82-8; 63,98-06-6; 64, 135-01-3; 65, 108-90-7; 66, 95-50-1.
Literature Cited (1) Kamlet, M. J.; Doherty, R. M.; Veith, G. D.; Taft, R. W.; Abraham, M. H. Enuiron. Sci. Technol. 1986, 20, 690. (2) Taft, R. W.; Abraham, M. H.; Doherty, R. M.; Kamlet, M. J. Nature (London) 1985, 313, 384. (3) Kamlet, M. J.; Doherty, R. M.; Abboud, J.-L. M.; Abraham, M. H.; Taft, R. W. J . Pharm. Sci. 1986, 75, 338. (4) Kamlet, M. J.; Abraham, M. H.; Doherty, R. M.; Taft, R. W. J. Am. Chem. SOC.1984,106,464. (5) Taft, R. W.; Abraham, M. H.; Famini, G. R.; Doherty, R. M.; Kamlet, M. J. J . Pharm. Sci. 1985, 74, 807. (6) Abraham, M. H.; Kamlet, M. J.; Taft, R. W. J. Chem. Soc., Perkin Trans. 2 1982, 923. ( 7 ) Taft, R. W.; Abraham, M. H.; Doherty, R. M.; Kamlet, M. J. J. Am. Chem. SOC.1985,107, 3105. (8) Hildebrand, J. H.; Scott, R. L. The Solubility of Nonelectrolytes, 3rd ed., Dover: New York, 1964.
Environ. Sci. Technol. 1987, 27, 155-162
Kamlet, M. J.; Carr, P. W.; Taft, R. W.; Abraham, M. H. J. Am. Chem. SOC.1981,103,6062. Kamlet, M. J.; Abboud, J.-L. M.; Taft, R. W.Prog. Phys. Org. Chem. 1981,13,485. Kamlet, M. J.; Abboud, J.-L. M.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983,48,2877. Taft, R. W.; Abboud, J.-L. M.; Kamlet, M. J.; Abraham, M. H. J. Solution Chem. 1985, 14, 153. Abboud, J.-L. M.; Sraidi, K.; Guiheneuf, G.; Negro, A.; Kamlet, M. J.; Taft, R. W. J. Org. Chem. 1985, 50, 2870. Kamlet, M. J.; Doherty, R. M.; Abraham, M. H.; Abboud, J.-L. M.; Taft, R. W. CHEMTECH 1986,16, 566. Kamlet, M. J.; Taft, R. W. Acta Chem. Scand., Ser. B 1985, B39, 611. Kamlet, M. J.; Abraham, D. J.; Doherty, R. M.; Taft, R. W.; Abraham, M. H. J. Pharm. Sci. 1986, 75,350. Reid, W. D. Experientia 1972, 28, 1058. Lipnick, R. L.; Johnson, D. E.; Gilford, J. H.; Bickings, C. K.; Newsome, L. D. Environ. Toxicol. Chem. 1985,4,281. Hansch, C.; Dunn, W. M. J. Pharm. Sci. 1972, 61, 1. Juhnke, V. I.; Ludemann, D. 2.Wasser Abwasser Forsch. 1978, 11, 161. Lipnick, R. L.; Dunn, W. J. In Quantitative Approaches to Drug Design; Proceedings of the Fourth European Symposium on Chemical Structure-Biological Activity:
(22) (23) (24) (25) (26)
Quantitative Approaches, Bath, U.K., Sept 6-9, 1982; Deardon, J. C., Ed.; Elsevier: Amsterdam, 1983; p 265. Kamlet, M. J., Naval Surface Weapons Center,unpublished results. Hammett, L. P. Physical Organic Chemistry;McGraw-Hill: New York, 1940; p 21. Franks, N. P.;Lieb, W. R. Nature (London)1984,310,599. Perutz, M. F. J . Cryst. Growth 1968, 2, 54. Farnell, K. J.; McMeekin, T. L. Arch. Biochem. Biophys.
1973,158, 702. (27) Abraham, D. J.; Perutz, M. F.; Phillips, S. E. V. ROC. Natl. Acad. Sci. U.S.A. 1983,80, 324. (28) Abraham, D. J.; Kennedy, P. E.; Mehanna, A. S.; Patwa, D. C.; Williams, F. L. J. Med. Chem. 1984, 27, 967. (29) Schoenborn,B. P. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 4195. (30) Helmer, F.; Kiehs, K.; Hansch, C. Biochemistry 1968, 7, 2858. (31) Leo, A.; Hansch, C.; Church, C. J. Med. Chem. 1969, 12, 766.
Received for review December 27, 1985. Accepted September 11,1986. The work by M.J.K. and R.M.D. was done under Naval Surface Weapons Center Independent Research Task IR-060.
Equilibration of Polychlorinated Biphenyls and Toxaphene with Air and Water Thomas J. Murphy,*,+Mlchael D. Mullin,t and Joseph A. Meyert
Chemistry Department, DePaul University, Chicago, Illinois 60604, and U.S. Environmental Protection Agency Large Lakes Research Station, Grosse Ile, Michigan 48138 The vapor pressure and solubility (as subcooled liquids) and Henry's law constants (HLCs) for the individual chlorobiphenyl compounds (CBC) in Aroclor 1242,1254, and 1260, as well as the HLCs for toxaphene, have been determined with an equilibration technique. The results were obtained from one set of measurements, for all of the CBC present in the materials of interest, the subcooled organic mixtures that are partitioning in the environment. to 5 The HLCs of the CBC varied from about 3.3 X X atmm3/mol at 20 "C, and those of the Aroclors were about 2 X 10" atmm3/mol. These HLCs are such that the transport rate of these compounds through the air/ water interface should be dependent on both the gas- and liquid-phase mass-transfer coefficients. The solubility of the polychlorinated biphenyls (PCBs) in water is shown to be significantly lower when a solution is prepared by adding an organic solution (acetone, methanol, etc.) of the PCBs to water. These results affect the design and the interpretation of the results from toxicity experiments. Introduction Polychlorinated biphenyls (PCBs) and toxaphene are each mixtures of a large number of individual compounds that are widely distributed in the environment. Their transport through the environment, while reasonably well understood, is quite complex and has not yet been quantified. The transport is controlled by the solubility, vapor pressure, and mass-transfer coefficients of the individual compounds in the mixtures. The major problem when this project was undertaken was that the values of these properties, and their range, either are not known or are DePaul University.
U S . Environmental Protection Agency Large Lakes Research
Station.
0013-938X/87/0921-0155$01.50/0
not known with sufficient accuracy (1-3). Without this information, good calculations of the fugacities of these compounds in different phases, and the direction and magnitude of the transport between phases, which is crucial for for an understanding of their environmental chemistry, cannot be made. Two different approaches have been taken to determine the physical properties of the Aroclor mixtures. The first is to make measurements on individual chlorobiphenyl compounds (CBC) and then to sum and extrapolate the results to the mixtures. The second approach is to make direct measurements on the commercial mixtures, the Aroclors and on mixtures found in the environment. The advantage of working with the individual compounds is that they can be obtained quite pure and measurements can be made more rapidly, accurately, and precisely. In recent years a number of studies on the solubility (4-6), vapor pressure (7-10))and Henry's law constant (HLC) (11-13) of individual CBC have been reported. There are two disadvantages of this approach. The first is that measurements have to be made on a large number of individual compounds. The second is that the individual CB and chlorocamphene compounds are only laboratory curiosities and are mostly solids but they occur in the environment only as mixtures of subcooled liquids. Corrections to the values measured on the solids then have to be made. These corrections can be greater than a factor of 30 for solubilities. The assumptions and errors introduced in making the corrections decrease the acctnacy of the final results. The advantages of making the measurements on the mixtures are that one measurement gives the desired information on all of the CBC present and that you are making the measurements on the material of interest, the mixtures. If the properties of the individual components
0 1987 American Chemical Society
Envlron. Sci. Technol., Voi. 21, No. 2, 1987 155
