Environ. Scl. Technol. 1986, 20, 690-695
Solubility Properties in Polymers and Biological Media. 7. An Analysis of Toxicant Properties That Influence Inhibition of Bioluminescence in Photobacterium phosphoreum (The Microtox Test) Mortlmer J. Kamlet* and Ruth M. Doherty
Naval Surface Weapons Center, White Oak Laboratory, Silver Spring, Maryland 20903-5000 Gilman D. Velth
Environmental Research Laboratory-Duluth,
U S . Environmental Protection Agency, Duluth, Minnesota 55804
Robert W. Taft
Department of Chemistry, University of California, Irvine, California 927 17 Michael H. Abraham
Department of Chemistry, University of Surrey, Guildford, Surrey GU2 5XH, United Kingdom
w Inhibition of bioluminescence in Photobacterium phosphoreum (the Microtox test) has been proposed as a cost-effective prescreening procedure to eliminate the relatively more inocuous chemicals from testing programs for toxicities of organic chemicals to fish. The biological response, as a function of toxicant properties, is given by
+
log ECSO(in pmol/L) = 7.61 - 4.11P/100 - 1.54a* 3.948 - 1.51am n = 38, r = 0.987, SD = 0.28 where P is the solute molar volume and a*,8, and a, are the solvatochromic parameters that measure dipolarity/ polarizability, hydrogen-bond acceptor basicity, and hydrogen-bond donor acidity of the solute (toxicant). The above equation applies to compounds that act by a nonreactive toxicity mechanism, and it is suggested that for certain compounds,which are outliers relative to the above equation, reactive toxicity properties mask the effects of the nonreactive mechanism. The above equation is compared with a correlation of log ECSOwith octanol/water partition coefficients. ~~
In the present report we explore the relationship of solute properties, expressed in terms of the solvatochromic parameters, to inhibition of bioluminescence in Photobacterium phosphoreum by organic nonelectrolyte toxicants. The experimental methods have been described in the standard procedure of the Beckman Microtox assay system ( I ) , which has been proposed as a cost-effective prescreening procedure to eliminate the relatively more inocuous chemicals from testing programs for toxicities of industrial pollutants and other organic chemicals to fish (2). The bioassay is based on the production of light by living luminescent bacteria, which is a reflection of the rate at which a complex set of energy-producing reactions is operating. Chemical inhibition of the enzymes involved will alter this rate, and subsequently the amount of light produced. The end point of the test is the concentration of toxicant needed to reduce light production by 50% after 5 min (EC,,) with reference to a standard emission from a toxicant-free solution. Curtis and co-workers (3) have reported fair correlation of Microtox test results with 96-h values for toxicity to the fathead minnow. The work described here is part of a continuing series of investigations wherein we hope to demonstrate that many disparate physicochemical, biochemical, toxicological, and pharmacological properties that depend on solute/solvent interactions and aqueous solubilities can be 690
Environ. Sci. Technoi., Vol. 20, No. 7, 1986
correlated, rationalized, and predicted by means of a single generalized linear solvation energy relationship of simple and conceptually explicit form. In earlier reports, we have pointed out that solubility properties, SP, of nonelectrolyte (4-8) and electrolyte solutes (9) in a variety of media can be expressed by linear combinations of terms that estimate the contributions of an endoergic cavity forming process and exoergic solute/solvent dipolar and hydrogen-bonding interactions. The endoergic cavity term measures the free energy or enthalpy input necessary to separate the solvent molecules and provide a suitably sized cavity for the solute. By use of the convention that subscript 1in the solvatochromic equations applies to the solvent and subscript 2 to the solute, the solute property that influences this term is its molar volume, P2,taken here as its molecular weight divided by its liquid density at 20 O C . The complementary solvent parameter is (6H2)1, the square of the Hildebrand solubility parameter (10, 11). The dipolar term, which measures the (usually) exoergic effects of solute/solvent dipole/dipole, dipole/induced dipole, and dispersion interactions, depends on a*l and a* being the solvatochromic parameter that scales molecular dipolarity/polarizability (12-14). For hydrogen bond acceptor (HBA) solutes in hydrogen bond donor (HBD) solvents like water, the exoergic hydrogen-bonding a and 8 being the solterm is measured by al and vatochromic parameters that measure HBD acidity and HBA basicity, and the subscript m indicating that, for amphihydrogen bonding compounds, the “monomer” value, applicable to the non-self-associated solute (7,151, is used. For strong HBD solutes in HBA solvents, hydrogen-bonding interactions may also lead to a term in (a,)g and P1. Accordingly, the generalized equation for solubility and solubility-rdated properties takes the form of eq 1. When SP = SPO+ A ( ~ H ~ )+I V Br*1~*2 ~ + Ca1(8m)2 + 0 P l ( a r n ) 2 ( 1 ) dealing with solubility properties of multiple solutes in single solvents, or with distributions between pairs of solvents, the factors relating to the solvent(s) may be subsumed in the constants in eq 1, and the dependence of each term in the resulting eq 2 is explicitly on the solute parameters. (We use v2/100 in order that the scale of the SP = SPo + mV2/100 + sa*2 + ~ ( a , )+~b(P,), (2) cavity term should be similar to those for
0013-936X/86/0920-0690$01.50/0
a2,and p2,
0 1986 American Chemical Society
which makes easier the evaluation of the relative contributions of the various terms to SP.) A high precision correlation by eq 2 (n = 105, r = 0.994, SD = 0.14) provided the first identification and evaluation of the solute physicochemical properties that influence solubilities of liquid aliphatic nonelectrolytes in water. The leading terms were found to be the exoergic effect of hydrogen bonding by HBD solvent water to HBA solutes (bD2)and the opposing endoergic cavity term (mv2/100) (6, 7). Similarly, a correlation by eq 2 of octanol/water partition coefficients of 102 liquid aliphatic and aromatic non-hydrogen bonding, HBA, and weak HBD solutes had the same leading terms and again showed excellent statistical goodness of fit (r = 0.989, SD = 0.18) ( 4 , 5). It has been shown in numerous studies (14) that purely chemical properties like water solubilities, S,, and octanol/water partition coefficients, KO,,correlate well with pharmacological and toxicological properties of those chemicals. The goodness of the relationship of toxicological properties to S, and KO,is dependent on the extent to which they tend to covary with the same changes in properties of the toxicant molecules. Many forms of toxicity are dependent on the pharmacokinetics and distribution of chemicals within the organism. Increasing water solubility (hydrophilicity) slows the passage of chemicals through membranes and favors excretion of the solute (in urine or through solution in the water passing through the gills), thus lessening the tendency to build up to toxic concentrations. Increasing lipophilicity (hydrophobicity), which has been considered to covary with KO,,leads to easier passage through membranes and greater distribution to hydrophobic areas of the organism. Consequently, in many studies of nonelectrolyte toxicants, the toxicity or biological activity of the chemical has been shown to vary inversely with water solubility and directly with KO,. It is particularly important in the latter regard that octanol and the protein and lipid components of the biosystems are stronger hydrogen bond acceptor bases than water: HBA base water solvent octanol solvent lipid R-CO-0-R protein R-CO-NH-R
P 0.2-0.4 0.8 0.5 0.8
For this reason, increasing a, should lead to increased partition into both octanol and the biosystems, and hence to increased toxicity. We have been particularly interested in the quantitative aspects of these latter effects, because we have found that many toxicological QSAR correlations with log KO,have tended to deteriorate in terms of statistical goodness of fit when phenolic solutes were included in the correlations. It is necessary to emphasize that two distinct types of toxicological mechanisms, reactive and nonreactive will be discussed here. The correlations presented here do not apply to reactive toxicity mechanisms, i.e., those which involve specific reactions such as Schiff-base formation between aldehyde toxicants and enzyme amine groups, or Michael addition of enzyme sulfhydryl groups to conjugated double bonds, as with acrolein or methyl vinyl ketone (17)[or unsaturated alcohols that can be oxidized in vivo to conjugated carbonyl compounds according to Lipnick’s “proelectrophile” mechanism ( I S ) ] . They refer, rather, to nonreactive toxicity behavior, wherein toxic effects can be shown by any organic nonelectrolyte if present in sufficient concentration, and wherein pharmacokinetic factors like partitioning and transport are rate controlling. As with most nonreactive
toxicological QSARs of simple molecules (16),the mechanism is usually considered to involve inhibition of physiological activity by saturation of lipophilic membranes, leading to hindrance of electrolyte transport across those membranes, and hence to anesthesia, narcosis, and, in the extreme, death [see, however, an opposing view (21)].
Results and Discussion Table I presents Microtox Test ECm results for 44 aliphatic and aromatic non-hydrogen bonding, HBA, HBD, and amphihydrogen bonding solutes (toxicants) for which values of V/100, a*,p, and a, are known or can be estimated. The same “ground rules” are applied to these data as were set forward earlier (5, 7) for correlations of log KO, and log S,, namely, (a) we add 0.10 to 9 / l O O of alicyclic and aromatic compounds, (b) in contradistinction to chloroaliphatic solvents, for which PI = 0.00, we use a p2 value of 0.10 for chloroaliphatic solutes, and (c) we use a a*, value of 0.40 for all monomer alkanol soluties, and 0 , values of 0.42 methanol, 0.45 for all primary alkanols, 0.51 for all secondary alkanols, and 0.57 for all tertiary alkanols. Further, as with the chloro compounds, but unlike the other non-self-associatingcompounds, we have found that the effective p2 value of pyridine solute is different from that of pyridine solvent (& = 0.64). Thus, we have found that a p2 value of 0.47 correctly predicts the octanol/water and cyclohexane/water partition coefficients and water solubility of pyridine solute, and we have used that p2 value in the present correlations. (We have also found that log K, and log S, values of a number of substituted pyridines accommodate well to the approximation p2 = PI - 0.17.) The ECMdata are mainly those of Curtis and co-workers (3) but include also some results reported by Hermens (19) and by De Zwart and Slooff (2). Values of EC50 for six compounds are given by both Curtis et al. and Hermens; the differences between the two sets of results ranged from 0.01 to 1.12 log units (for CH,CCl3). The average difference of 0.27 log unit for the other five common solutes gives an indication of the usual reproducibility of the measurements, and the conflicting results for CH3CC13show how vulnerable the calculational method is to the occasional very much out-of-line result. Before proceeding with the correlations, we peremptorily excluded two toxicants, acetaldehyde and propionaldehyde from the correlations on the basis that they most probably acted by an additional specific reactive toxicity mechanism. Our reasoning was as follows: Alcohols and corresponding carbonyl compounds have similar values of 9, a*,and /3 (or B,), and hence similar solubility properties. Thus, as is shown in Table 11, solubilities in water and octanol/water partition coefficients are similar for acetone and 2-propanol, 2-butanone and 2-butanol, acetaldehyde and ethanol, and propionaldehyde and 1-propanol. Mirroring this trend and characteristically of nonreactive toxicological behavior, the log ECm values are similar for acetone and 2-propanol and for 2-butanone and 2-butanol (the latter value being estimated from the 1-butanol EC50 value and other secondary alcohol/primary alcohol differences). In the cases of acetaldehyde and propionaldehyde, however, the log ECMvalues are 2.0 and 2.8 log units lower than for ethanol and 1-propanol. This is characteristic of reactive toxicological behavior and suggests that the toxic effects of these aldehydes are not controlled by general solubility and transport properties but rather that a specific chemical interaction is involved. Further, the effect is as anticipated. Aldehydes would be expected to react rapidly and irreversibly with amine groups that are omnipresent in biological systems (to form Schiff bases, Environ. Sci. Technol., Vol. 20, No. 7, 1986
691
Table I. Data Used in Correlation of Microtox Test Results log EC,, pmol/L ref 3 av calcdCeq 4
no.
toxicant
v/100"
T*"
P
1 2 3 4
methanol ethanol 1-propanol 2-propanol 1-butanol 2-methyl-1-propanol 3-pentanol I-hexanol 1-heptanol 1-octanol 2-decanol acetone 2-butanone 4-methyl-2-pentanone 2-octanone ethyl acetate ethyl propionate diethyl ether di-n-butyl ether dimethylformamide CH,CCl, CHCl=CCl, CICHzCHzCl CHClzCHClz benzene toluene o-xylene chlorobenzene 1,3-dichlorobenzene 1,2,3-trichlorobenzene l-CH3-3,4-CsH3ClZ phenol o-cresol 4-t-Bu-CGH40H 2,4-dimethylphenol 4-nitrophenol pyridine 6-methyl-5-hepten-2-one
0.405 0.584 0.748 0.765 0.915 0.920 1.073 1.256 1.414 1.575 1.907 0.734 0.895 1.253 1.563 0.978 1.146 1.046 1.694 0.774 0.996 0.897 0.787 1.052 0.989 1.169 1.329 1.118i (1.226)j [1.33411 [1.437]1 (0.989)j (1.163)j (1.698)j (1.345)j [1.1501j 0.905' 1.476
0.40e 0.40e 0.40' 0.40' 0.40' 0.40' 0.40' 0.40' O.4Oe 0.40' 0.40' 0.71 0.67 (0.65) (0.65) 0.55 0.47 0.27 0.24 0.88 0.49 0.53 0.81 0.95 0.59 0.54 0.47 0.71 (0.80) (0.85) (0.75) (0.75Ik (0.75) (0.75) (0.75) (1.17)k 0.87 (0.70)
0.42f 0.46 0.45f 0.511 0.46 0.46 0.51f 0.46 0.46 0.46 0.51f 0.48 0.48 (0.48) (0.48) 0.45 0.46 0.47 0.46 0.69 0.10h 0.10h 0.10h 0.10h 0.10 0.11 0.13 0.07 (0.03) (0.03) (0.07) 0.33"' (0.37) (0.37) (0.41) (0.52)" 0.47' (0.48)
0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0 0 0 0 0 0 0 0 0 0 0 0 0.13 0 0 0 0 0 0 0 0.61 (0.50) (0.58) (0.50) 1.00 0 0
41 42
cyclohexanol cyclohexanone 5-methyl-2-hexanone 2-decanone
1.140' 1.135' 1.286 1.894
0.40' 0.75 (0.65) (0.63)
0.51f 0.53 (0.48) (0.48)
Outliers 0.33 0 0 0
43 44
acetaldehyde propionaldehyde
Known To Act by Additional Specific Toxicity Mechanism 0.562 (0.67) 0.42 0 3.88 3.88 0 2.35 2.35 (0.63) 0.42 0.721
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 33 34 35 36 37 38 39 40
ref 19
amb
6.12 5.16 4.58 4.23 2.82 1.93 1.56 5.56
4.88 ((1.78))i 3.16 4.05 3.31 2.29 1.94 2.12 1.35 1.14 0.94
6.50 5.98
6.36 5.98 5.16 5.76 5.76 4.49 4.54 4.35 4.35 4.23 2.59 2.71 1.93 1.69 1.62 0.87 0.87 5.57 5.57 4.85 4.85 2.90 2.90 2.14 2.14 4.848 3.848 4.88 2.68 2.68 5.43 5.43 2.90 2.90 3.16 4.05 1.70 1.70 3.31 2.29 1.94 2.12 1.35 1.14 0.94 2.63 2.63 2.288 0.15 0.15 1.55 1.55 1.97 1.979 4.518 2.14 2.14
6.47 5.86 5.18 5.35* 4.50 4.48 4.09 3.10* 2.45* 1.79 0.66 5.38 4.78 3.34* 2.06 4.51* 3.98 4.74 2.08** 5.78* 3.15 3.49* 3.51* 2.01* 3.02* 2.42 1.93 2.19 1.45 0.92 0.81 2.76 2.37 0.05 1.78 1.61* 4.39 2.34
3.06 2.28 3.93 1.70
3.77** 3.87***** 3.20** 0.73***
3.06 2.28 3.93 1.70
logd KO,
log ECm; calcd from eq 5a
-0.70 -0.25 0.28 0.05 0.72 0.75 1.21 2.03 2.41 2.97
5.88' 5.43* 4.91 5.14 4.48 4.46 3.99 3.18* 2.80** 2.24
-0.24 0.29
5.42 4.90
0.73 1.21 0.89
4.47* 3.99 4.30*
-1.01 2.49 2.29 1.48
6.18 2.72 2.92 3.72
2.13 2.69 3.20 2.84 3.53 4.20 3.98 1.41 1.95 3.31 2.30 1.91 0.66
3.08 2.52 2.02 2.37 1.69 1.03 1.24 3.74*** 3.25** 1.91***** 2.91**** 3.30*** 4.53
1.23 0.81
3.97** 4.39******
" Values in parentheses are estimated from corresponding values for closely related compounds. Values in brackets are particularly uncertain. *Details regarding the amscale of "monomer" HBD acidities (applicable to amphihydrogen bonding compounds acting as nonself-associated solutes) will be reported in a future paper. Single asterisk denotes deviation from correlation equation by more than one standard deviation, double asterisk by more than two standard deviations, etc. dReference 23. 'Values are for ?r*, (estimated from a **/dipole moment correlation (25)). fValues are for brn(5,7). #Reference 2. hAs set forth in ref 5 and 7, we use p = 0.10 for chloroaliphatic solutes but continue to use p = 0.00 for chloroaliphatic solvents. 'We have ignored this obviously out-of-line result. 'As set forth in ref 7, we add 0.10 to v/lOO of aromatic and alicyclic compounds. kEstimated from dipole moments. mEstimated from gas-phase basicities. "Summation of estimated p values at both HBA sites. "The & value of 0.47 is different from p1 = 0.64 for pyridine solvent; see text. 9This result indicates lesser toxicity than might be the case if solute dissociation were repressed. RCH=N-R'), and wherever aldehyde toxicities have,been compared with predictions from nonreactive toxicological QSARs, similar enhanced toxicities have been observed. Thus, we have found that acetaldehyde also shows greater than predicted toxicity to the fathead minnow, as do butyraldehyde, hexanal, and acrolein, and acetaldehyde, butyraldehyde, and benzaldehyde show greater than predicted toxicity to the golden orfe fish. The multiple linear regression equation for the other 42 toxicants of Table I is given by eq 3, log EC60 = (7.45 f 0.36) - (3.93 f 0.2O)Vz/lOO (1.58 f 0.34)~*2+ (3.80 i 0.41)& - (1.53 f n = 42, r = 0.966, SD = 0.43 (3) O.31)(a,J2 692
Environ. Sci. Technol., Vol. 20, No. 7, 1986
where EC,, is expressed in micromoles per liter. By the standards that we have applied to other QSAR correlations by eq 2, if not necessarily by the standards usually applied to correlations of biological properties, the r and SD values for eq 3 represent unsatisfactory goodness of fit. We therefore undertook to examine the outliers in the correlation to ascertain whether nonconformance with eq 3 is more likely due to reactive toxicology behavior, as with the aldehydes, or incorrect experimental determinations, as with l,l,l-trichloroethane. The four most out of line results are for the following: cyclohexanone (compound 40 of Table I), for which A (observed minus calculated) = -1.59; cyclohexanol(39),A = -0.71; 5-methyl-2-hexanone (41), A = +0.73; 2-decanone (42), A = +0.97.
r
Table 11. Comparison of Solubilities and Toxicities of Alcohols and Corresponding Carbonyl Compounds
CH&H(OH)CH3 CH3COCH3 difference
log sw 0.83 0.92 -0.09
1% K o w 0.05 4.24 0.29
CH&H(OH)CH&H, CH3COCHZCHS difference
0.39 0.48 -0.09
0.61 0.28 0.33
CH3CHzOH CHSCH=O difference
1.10 1.19 -0.09
solute
CH3CHZCHZOH CH&H2CH=O difference
0.62 0.60 0.02
rl
r = O B 7 (EX DUlU€RS\
log ECm
5.76 5.57 0.19
A
4.70 (est)
4.85 -0.15
I
0
5.98 3.88 2.10 0.30 0.59 -0.29
..
I
5.16 2.35 2.81 1.0
It is reasonable to suggest that cyclohexanone (401, which is more toxic than predicted by more than five standard deviations of eq 3, and cyclohexanol, which may be oxidized to 40 in vivo, are outliers because they act by a reactive toxicological mechanism, similar to that for the aldehydes. Cyclohexanone is known to be more reactive with RNH, compounds than acyclic ketones; e.g,, rate of semicarbazone formation (R1R2C=0 + H~NNH-CO-NHP R1R2C=NNH-CO-NH, + H20) is 7 times greater than that for acetone and 63 times greater than that for 3pentanone (20). If 39 and 40 are excluded from the correlation (also justified for the latter on purely statistical grounds), n = 40, r = 0.982, and SD = 0.32. In the case of the other two outliers, 5-methyl-2-hexanone (41) and 2-decanone (42)) both less toxic than calculated, the out-of-line behavior is evident, but the reason is not. Thus, it is seen that the usual effect with increasing carbon number in a homologous series is toward lower ECa (by about 0.6-0.7 log unit per carbon atom); compare, for example, the alkanols 1-11, the ketones 12-15, and the aromatics 25-29. In going from 4-methyl-2-pentanone (14) to 41, however, the reported EC50 value increases rather than decreases, and on going from 2-octanone (15) to 42, the decrease in log EC50 is about 0.8 log unit less than would be expected from other A log ECw/Anc results. On this basis, we believe that the behavior of 41 and 42 reflect either experimental error or an unconsidered specific relationship between solute dimensions and the size and shape of a receptor pocket in an enzyme, as discussed by Franks and Leib for inhibition of activity of firefly luciferase by general anesthetics (21). Although the correlation to be preferred on purely statistical ground would be for n = 41, excluding only cyclohexanone, we consider that the quantitative structure-activity relationship to be preferred on biochemical grounds (Le., that which best reflects the relative contributions of the cavity, dipolarity, and hydrogen-bonding terms to Microtox test results) is one wherein the four outliers previously discussed are excluded. The multiple linear regression equation for compounds 1-38 of Table I is given by eq 4. A plot of eq 4 is shown in Figure 1 log ECB0(in pmol/L) = (7.61 f 0.25) - (4.11 f 0.14)~2/100- (1.54 z t 0 . 2 3 ) ~ "+~(3.94 f 0.28)& (1.51 f 0.21)(a,,J2 n = 38, r = 0.987, SD = 0.28 (4)
-
It is seen that the only significant difference between eq 3 and 4 is in the statistical goodness of fit. It is also seen in eq 3 and 4 that increasing solute molar volume, increasing solute HBD acidity, and decreasing solute HBA basicity, all of which lead to lower solubility in water, lead to greater toxicity (lower EC50 values) of the toxicants but
20
-
4.0
3.0
6.0
80
-
7.61 4.iilim I M ~t* WB.1.51am
Flgure 1. Correlation of Microtox test results by eq 4. Note that the
phenol derivatives fit the correlation quite well.
that higher solute dipolarity/polarizability ( T * ~ which ) leads to greater solubility in water also leads to lower ECM values. We have seen similar converse effects of solute dipolarity on toxicities to the fathead minnow, golden orfe, and Madison 517 fungus and on tadpole narcosis, and we feel that they may have important implications regarding the mechanism of toxicity or narcosis, as will be discussed in a future paper. It is also seen in eq 3 and 4 that, as with water solubility (4,5) and odanol/water partition ( 2 , 3 ) ,the largest effects on toxicity are the opposing influences of solute HBA basicity and molar volume. Unlike S, and KO,,however, ~ (a& are also important contributors the terms in T * and to the observed effects. We have discussed in detail the out-of-line results in Table I. Also worthy of mention are several results that do fit eq 4, where other considerations might suggest that they should not. We have found that when eq 2 is applied to the correlation of toxicities of narcotic industrial chemicals to either the golden orfe fish or the fathead minnow, there are trends in both instances for seven or eight carboxylic esters to be too toxic by as much as 2.1 log units. In the case of the golden orfe, the enhanced toxicity correlates well with neutral or alkaline hydrolysis rates (22). In contradistinction to these findings, however, ethyl acetate (16) and ethyl propionate (17) appear to fit eq 4 reasonably well. Such possible differences in i n vivo behavior might limit the applicability of the Microtox test to prediction of f i s h kill results for certain classes of compounds but might also point to interspecies differences in the operation of specific toxicity mechanisms.
Correlationswith Octanol/ Water Partition Coefficients. Octanol/water partition coefficients have been used successfully in hundreds of QSAR studies involving toxicological properties like those considered here (23). The success of these correlations is inextricably linked to the extent to which the molecular factors that influence log KO,also influence the biological response in the same way. Given this, one may draw some conclusions about the toxic mechanism and the factors that influence log KO, from the differences in goodness of fit between log K , and the toxicological end point as different classes of compounds are considered. Hermens (29)has reported near unit slope, r = 0.953, and SD = 0.53 in a correlation of his results (n = 23) with log Kow. The goodness of fit, however, is strongly dependent on the classes of compounds that are included in Environ. Sci. Technol., Vol. 20, No. 7, 1986 693
I . 6. O t
I
I
\*.
0
5.0 -
8
4.0 -
M
s” 3.0 -
-1.0
I
I
I
0
1.0
2.0
,.
3.0
I
4.0
Log Kow
Figure 2. Mlcrotox test results plotted vs. octanol/water partltlon coefficients. Note that the phenol derivatives are more toxic than predicted by eq 5a (regression line corresponds to eq 5a).
the data set. Values of log KO,are known for 33 of the toxicants considered above and are included in Table I. If we first consider the 17 non-HBD solutes of Table I, the correlation is given by eq 5a. If the results for the 11 log EC50 = 5.19 - 0.99 log KO, n = 17, r = 0.977, SD = 0.34 (5a) log EC50 = 5.29 - 1.07 log KO, n = 28, r = 0.967, SD = 0.42 (5b) log EC50 = 5.21 - 1.13 log KO, n = 33, r = 0.935, SD = 0.61 ( 5 4 alcohol solutes are next included, the correlation is given by eq 5b. When, finally, the results for the five phenol solutes are included, the correlation is given by eq 5c. A plot of log ECm results against log KO,is shown in Figure 2. A comparison of the observed results with those calculated by eq 5a is also included in Table I. (Parenthetically, it deserves comment that the outliers in the correlation with the solvatochromicparameters are also outliers in the correlation with log Kow) It is seen in the equations, the plot, and the table that the correlation of log ECso with log KO,is quite good for the non-HBD solutes, that the goodness of fit falls off slightly when the weak HBD alcohol solutes are included, and that the correlation deteriorates seriously when the strong HBD phenolic solutes are included. We have seen this pattern of behavior, i.e., good to excellent correlations with log KO,for non-HBD and weak HBD toxicants and deterioration of correlation when strong HBD toxicants are included, in many QSARs involving toxicological properties. For comparison with the above situation, we have repeated the correlation by eq 2, excluding the data for the HBD solutes and the term in (am)2.The resulting multiple linear regression equation is given by eq 6. It is seen that log EC50 = (7.99 f 0.46) (4.11 f 0.26)Vz/lOO - (2.05 f 0.37)~*2+ n = 22, r = 0.982, SD = 0.30 (6) (3.84 f 0.31)& the coefficients of the independent variables are quite similar in eq 4 and 6 and that the goodness of fit did not suffer when the HBD solutes were included. We believe that the conformance of the phenolic toxicants to eq 4 and their nonconformance with eq 5a is a consequence of two effects. (a) The organism probably 694
Environ. Scl. Technol., Vol. 20, No. 7, 1986
responds to (am)z of the toxicant, whereas log KO,responds to A(am)Z,the difference in a, between the phenol and octanol. (b) The high T*, values of the phenolic toxicants favor distribution into water over octanol but also favor increased toxicity. The correlation with the solvatochromic parameters correctly reflects these effects; the correlation with log Kowdoes not. Finally, it is not a new realization that increasing dipolarity and HBD acidity substantially increase toxicities of the nonelectrolytes with respect to predictions from the lipid-partitioning models alone (24). Early observations of this effect led to proposals of separate mechanisms of narcosis for dipolar strong HBD compounds like the phenols. Indeed, unless the influences of the chemical properties are correctly unraveled and evaluated (as we hope we have done in the present study), the effect of increasing dipolarity within a series such as the phenols in Table I can be confused with the simultaneous increase in HBD acidity. Thus, this paper has shown that, among the narcotic chemicals, the phenols are more toxic than alcohols and nondipolar compounds of equal log K,,, not only because of greater HBD acidity but also because of their generally greater dipolarity. This finding is extremely important to the understanding of the mechanism(s) of narcosis and toxicity because it permits a single uniform model of narcosis, rather than an ensemble of models for narrowly defined classes.
Acknowledgments We are most grateful to Nicholas P. Franks and William Lieb of Imperial College (London) for extremely useful discussions and guidance. The work by R.M.D. and M.J.K. was done under Naval Surface Weapons Center Independent Research Task IR-060. M.J.K. dedicates his contribution to the present work to organic chemist Emanuel Lurie, who is not forgotten. Registry No. 1,67-56-1;2, 64-17-5; 3, 71-23-8; 4, 67-63-0; 5, 71-36-3;6,7843-1; 7,584-02-1;8,111-27-3;9,111-70-6;10,111-87-5; 11,1120-06-5; 12,67-64-1; 13,78-93-3; 14,108-10-1; 15,111-13-7; 16, 141-78-6; 17, 105-37-3; 18,60-29-7; 19, 142-96-1;20, 68-12-2; 21,7145-6; 22,79-01-6; 23,107-06-2; 24,79-34-5; 25,71-43-2; 26, 108-88-3;27, 95-47-6; 28, 108-90-7;29, 541-73-1; 30, 87-61-6; 31, 95-75-0; 32, 108-95-2; 33, 95-48-7; 34, 98-54-4; 35, 105-67-9; 36, 100-02-7;37,110-86-1; 38,110-93-0;39,108-93-0; 40,108-94-1;41, 110-12-3; 42, 693-54-9; 43, 75-07-0; 44, 123-38-6.
Literature Cited Beckman Instruments, Inc. “Operating Instructions MicrotoxTMAnalyzer Model 2055” Interim Manual 100697, Microbics Operations, Carlsbad, CA, 1979. De Zwart, D.; Slooff, W. Aquat. Toricol. 1983, 4 , 129. Curtis, C.; Lima, A.; Lozano, S.J.; Veith, G. D. ASTM Spec. Tech. Publ. 1982, No. 766, 170. Kamlet, M. J.; Abraham, M. H.; Doherty, R. M.; Taft, R. W. J,Am. Chem. SOC.1984,106,464. Taft, R. W.; Abraham, M. H.; Famini, G. R.; Doherty, R. M.; Kamlet, M. J. J. Pharm. Sci. 1985, 74, 807. Taft, R. W.; Abraham, M. H.; Doherty, R. M.; Kamlet, M. J. Nature (London) 1985, 313, 384. Kamlet, M. J.; Doherty, R. M.; Abboud, J.-L. M.; Abraham, M. H.; Taft, R. W. J . Pharm. Sci., in press. Abraham, M. H.; Kamlet, M. J.; Taft, R. W. J. Chem. Soc., Perkin Trans. 2 1982, 923. Taft, R. W.; Abraham, M. H.; Doherty, R. M.; Kamlet, M. J. J . Am. Chem. SOC.1985, 107, 3105. Hildebrand, J. H.; Scott, R. L. The Solubility of Nonelectrolytes, 3rd ed.; Dover: New York, 1964. 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.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983,48, 2877.
Environ. Sci. Technol. 1986, 20, 695-702
(13) Kamlet, M.J.; Abboud, J.-L. M.; Taft, R. W. Prog. Phys. Org. Chem. 1981,13,485. (14) Taft, R.W.; Abboud, J.-L. M.; Kamlet, M. J.; Abraham, M. H. J. Solution Chem. 1985,14, 153. (15) Abboud, J.-L. M.;Sraidi, K.; Guiheneuf, G.; Negro, A.; Kamlet, M. J.; Taft, R. W. J. Org. Chem. 1985,50,2870. (16) Hansch, C.; Dunn, W. M. J. Pharm. Sci. 1972, 61,1. (17) Reid, W. D. Experientia 1972,28, 1058. (18) Lipnick, R.L.; Johnson, D. E.; Gilford, J. H.; Bickings, C. K.; Newsome, L. D. Environ. Toxicol. Chem. 1985,4,281. (19) Hermens, J. Ph.D. Thesis, University of Utrecht, Utrecht, The Netherlands, 1983,p 39. (20) Hine, J. Physical Organic Chemistry;McGraw-Hill: New York, 1956;p 248.
(21) Franks, N. P.; Lieb, W. R. Nature (London) 1984,310,599. (22) Kamlet, M.J. Naval Surface Weapons Center, Silver Spring, MD, unpublished information. (23) Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and Biology; Wiley-Interscience: New York, 1979. (24) Veith, G. D.;Call, D. J.; Brooke, L. T. Can. J.Fish Aquat. Sci. 1983,40,743. (25) Abboud, J.-L. M.;Kamlet, M. J.; Taft, R. W. J. Am. Chem. SOC. 1981,103,1080.
Received for review May 6,1985.Revised manuscript received October 22,1985. Accepted March 12, 1986.
X-ray Photoelectric Spectroscopy Determination of a Conceptual Leaching Model of Retorted Oil Shale Jeffrey L. Feerer,+ Allen G. Miller,$and W. Fred Ramirez”t
Department of Chemical Engineering, University of Colorado-Boulder, Boulder, Colorado 80302 Two hypotheses are proposed to describe the leaching of solid wastes. One hypothesis involves formation of a leached zone around particles of solid waste in contact with water, resulting in a diffusion barrier to release of soluble pollutants. The other hypotheses relies on pore diffusion and surface reaction as the dominating mechanisms controlling leachate release. X-ray photoelectric spectroscopy (XPS) is used to help determine which model more closely describes leaching of retorted oil shale. Although some evidence of possible Al(OH)3 precipitation and relative depletion of sodium on the surface is found, a leached zone is apparently not formed on retorted oil shale during leaching.
Introduction Although leaching is a common industrial process, it is also a natural phenomenon in the environment whenever a soluble solid interacts with water. The contact of groundwater on solid waste, the effects of acid rain on soils, the dissolution of particulate atmospheric pollutants, and the solute chemistry of aquifers all depend on the nature of the leaching process. Leaching is described in a straightforward manner for rather simple industrial materials, yet little is understood concerning the fundamental mechanisms of leachate chemistry and transport in environmental settings. Materials that are leached in the environment are often either porous or crystalline aggregates or a mixture of codisposed solid wastes. Often these mixtures have components that are of differing behavior when contacted by water. Any one of several transfer mechanisms may control release of soluble species from the solid, depending on the chemicals present. Dissolution kinetics, mass transfer, ion exchange, pore diffusion, diffusion through the bulk solid, or precipitation may be important in describing the leaching process. All of these mass transfer or chemical rate processes are highly dependent on the composition of the solid surface. The key to understanding leaching in the environment lies in developing a concept as to the changing composition of the University of Colorado-Boulder. Corporation.
4 IBM
0013-936X/86/0920-0695$01.50/0
Boulder, Colorado 80309, and IBM Corporation,
surface of natural materials in contact with water, and then establishing a methodology for modeling the leaching behavior of complex mixtures. X-ray photoelectron spectroscopy (XPS) can be used to help develop a conceptual model of the leaching process in solid wastes since it is a highly surface-sensitive technique that will give clues as to the changes in surface composition and molecular structure during leaching. XPS is now a common surface analysis technique which has been reviewed often (1-3). One of the more definitive geochemical applications of X P S involved clarifying the dissolution mechanism of pure solids. The controversy revolved around the existence of either a “leached zone” or an amorphous precipitate at the surface of solid particles exposed to water. This surface layer would exist solely for kinetic reasons and would dissolve at the surface of each particle. Diffusion through this barrier would control the release of ions from the mineral upon prolonged contact with water (4-6). Support for this hypothesis came from the observation in many feldspar leaching studies that the solvent was strongly depleted in aluminum and silicon relative to the feldspar. However, Petrovic et al. (7) and others (8, 9) used XPS to show that a leached zone or a precipitate coating does not occur, and dissolution is apparently controlled by surface reaction of the unaltered mineral with the solution at the interface between these two phases. Berner and Holdren (10)demonstrated using SEM in conjunction with XPS that dissollution of pure minerals occurs selectively at surface sites of relatively higher energy such as dislocations. Fung and Sanipelli (11)subsequently found small aluminum silicate particles on the surface of leached feldspar, which accounted for the solvent being strongly depleted in aluminum and silicon. This secondary mineral phase always occurred as discrete particles and could not be construed as a diffusion barrier. Thus, a diffusion barrier has not yet been found on pure solids undergoing leaching. A mixture of porous particles of different solids, however, could form a leached zone upon contact with water. Some chemical species, such as strongly ionic salts, would dissolve faster than others, leaving a porous zone of insoluble material around and through which aqueous ionic species from the interior of the particle would diffuse. The
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