Environ. Sci. Techno/. 1995, 29, 1267-1272
QSAR Evaluation of a=Terthienyl Phototoxicity GILMAN D. VEITH,*,' O V A N E S G. M E K E N Y A N , I GERALD T. ANKLEY,+ AND DANIEL J. CALL* US.Environmental Protection Agency, Environmental Research LaboratoTDuluth, 6201 Congdon Boulevard, Duluth, Minnesota 55804, and Lake Superior Research Institute, University of Wisconsin-Superior, Superior, Wisconsin 54880
The concept that phototoxic chemicals can be identified in chemical risk assessments by computing the energy difference between highest occupied and lowest unoccupied molecular orbitals (HOMO-LUMO gap) was evaluated using t w o new sets of phototoxicity data from the recent literature. The original model, developed from data with unsubstituted PAH toxicity to Daphnia magna, showed that phototoxicity was observed when the HOMO-LUMO gap varied between 6.7 and 7.5 eV. All substituted a-terthienyls that were phototoxic to mosquito larvae and to brine shrimp had HOMO-LUMO gap energies within the 7.1 f 0.4 eV "phototoxicity window". The a-terthienyls within this range that did not exhibit phototoxicity contained carboxyl or other polar substituents, which likely prevented bioaccumulation in the organisms. Polyamino and polynitro derivatives of toluene in munitions wastes were reported to be phototoxic to sea urchins even though the HOMO-LUMO gap energies exceeded 8.0 eV. Because the same toluenes were not phototoxic to D. magna nor to Escherichia coli, w e suggest that the developmental effects observed in the sea urchins were caused by electrophilic species from metabolic activation rather than the production of oxygen radicals from photo induced excited states of the molecules.
Introduction The phototoxicity of chemicals is a function of molecular properties that affect their bioaccumulation in organisms, their ability to absorb sunlight, and the characteristics of their excited states (1-3). Of particular significance in environmental risk assessment are triplet states of the chemical which subsequently interact with molecular oxygen to create superoxide anion radicals and other reactive oxygen species (4-6). The production of cellular oxygen radicals causes cytotoxicity similar to other oxidative stress mechanisms (7, 8);however, phototoxicity is often so dramatic in destroying tissue and organisms that symptoms of conventional oxidative stress are likely to be observed only under chronic low-dose exposure. Because conventional hazard assessments typically are conducted in laboratorysettings,in the absence of Wlight, the risks of phototoxic chemicals under environmental conditions (Le., in sunlight) can be underestimated by orders of magnitude. Moreover, in the presence of natural sunlight, large bioconcentration factors can significantly increase the toxicity of even slightly phototoxic chemicals. The greatest environmental risks are likely be associated with species (e.g., amphibians) whose life cycles or styles expose them to phototoxic chemicals in contaminated sediments, where significant biaccumulation can occur, and then to sunlight where toxicity may be induced (9,10). Identificationof potentially phototoxic chemicals is not simple. Predictions based on the behavior of molecular analogues is easily confounded by examples such as anthracene, which is highly phototoxic while its nonlinear analogue phenanthrene is not toxic. Equally important, the high cost of bioassays generally precludes attempts to identify the most hazardous chemicals from among the hundreds of potentiallyphototoxic chemicals in commerce. We hope to solve this problem by developing structureactivity relationships that accurately identify potentially phototoxic chemicals and focus valuable testing resources on the chemicals posing the greatest phototoxicityhazards. The well-establishedrelationship between bioconcentration potential and 1-octanollwaterpartition coefficient seems to be adequate for eliminating many nonaccumulating chemicals from consideration (11). However, even with this level of initial screening, potentially bioaccumulative chemicals form a class that numbers well over 10 000 chemicals (12). Building upon the mechanistic work of Giesy and Newsted (11,we recently developed a method of identifylng potentiallyphototoxic chemicals from chemical structure (3). Briefly, we proposed that phototoxicity was a function of light absorption both in terms of wavelength energy and intensity, the energy and stability of the triplet state of the molecule, and the persistence of the ground-state molecule with respect to decomposition or other reactions in the ambient environment. We reasoned that each of these processes could be approximated from individual relationships with the energy difference between the highest occupied (HOMO) and lowest unoccupied (LUMO) mo* To whom correspondence may be addressed. + U S . Environmental Protection Agency. +
0013-936W95/0929-1267$09.00/0
D 1995 American Chemical Society
Lake Superior Research Institute.
VOL. 29. NO. 5, 1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY
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F (2)
I
I
3’ 4‘ FIGURE 2. a-Terthienyl framework with numbers indicating the points of substitution.
I
,8
0.3
8.5
9
HOMO-LUMO Gap [ey
FIGURE 1. Variation of PAH phototoxicity with HOMO-LUMO gap and the phototoxicity window.
lecular orbitals,commonlycalled the HOMO-LUMOenergy gap (13). Consequently,we plotted the phototoxicity data for PAHs versus HOMO-LUMO energy gap and found the expected parabolic relationship (see Figure 1). At the large HOMO-LUMO gap energies commonly found inaliphatic or slightlydelocalizedchemicals,sunlight absorption and triplet-state formation are minimal. With decreasing HOMO-LUMO gap energies, molecules begin absorbing the more energetic wavelengths in sunlight present in the W - B and then W - A regions. An optimum HOMO-LUMO gap for phototoxicityof UnsubstitutedPAHs was found at 7.1 k 0.4 eV (3). At smaller values of gap energy,the phototoxicity decreased from the optimum due, in part, toinstabilityoftheparentsuuctureandtheirexcited states. We called the range of HOMO-LUMO gap energy where phototoxicity in Daphnia magna was observed the “phototoxicitywindow”. The phototoxicity window unambiguouslydiscriminated PAHs that are highly phototoxic from those that are not. We hypothesized that the combined use of structureactivityrelationshipsfor hioaccumulation and phototoxicity may be a desirable addition to initial risk assessment protocols. However,further evaluation concerning several issues ofthe approach is needed. First, the computational chemistry involved in such analyses is quite extensive for large molecules. It is unlikely that 50 large chemicals per week could be thoroughly screened even with highperformance computing. Moreover, our initial identification of the phototoxicity window was developed from the only high-quality phototoxicity data available in the literature, which, despite the elegant experimentsto measure true phototoxicity, unfortunately included only unsubstituted and inflexible PAHs tested with a single aquatic species. The purpose of this workis to further evaluate the phototoxicity window concept with some a-terthienyl derivatives thought to be phototoxic to mosquito larvae and with munition waste constituents also thought to he phototoxic to aquatic organism.
Experimental Methods We used the data of Marles et al. (14) involving 34 a-terthienyl derivatives and light and dark toxicity tests with larvae of the mosquito Aedes atropalpus and nauplii of the brine shrimp Artemia salina. These experiments were conducted using only W - A light over the range of approximately320-400nm. Males et al. originallyreported data for three other a-terthienyls and four a-thiophene derivatives of benzene, naphthalene, and pyridine. How1268. ENVIRONMENTAL SCIENCE &TECHNOLOGY I VOL. 29,
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ever, the authors observed rapid photodegradation with these chemicals, resulting in greater toxicity in the dark than in the presence of W light. Consequently, the data derived for these seven chemicals were removed from our analysis. The structure of a-terthienyl is presented in Figure 2. The derivatives tested are presented in Table 1 together withthedark/light toxicityLC50ratio.whichwascomputed by dividing the LC50 for the chemical without light by the LC50 in the presence of light. As was true for certain of the PAHs investigated by Newsted and Giesy (1). some of the chemicalshave no measurable LC50 or at least have a LC50 greater than the water solubility under the chosen test conditions. Table 1 contains numerous examples of “nontoxic” chemicals that become extremely toxic in the presence of light, and the dark/light toxicity ratio is expressed as a “greater than” ratio. While these data preclude strict quantitative interpretation, they are extremelyuseful nonethelessinevaluatingtheconceptofthe phototoxicity window. We also used the data of Davenport et al. (19,who reported the light-enhanced toxicity of common constituents of munition wastes to embryos of the sea urchin Lytechinus uariagatus. The chemicals were nitrotoluenes and toluidines (Table 1) that were tested in the presence of a W - A light, with a filter to remove all light below 312 nm. The sea urchin test results were highly qualitative in that positive effects were reported with an increasing numberof“+”,indicatinganincreaseinthedegreeofeffects on embryo development. The differences in the number of between dark and light experiments were converted to the relative toxicity values we report in Table 1. HOMO-LUMO gap energies for all chemicals were computed on a VAX 4500 computer using the PM3 Hamiltonian within the public version of MOPACG (16). The electronic parameters are normally quantified after the chemical structure is optimized to minimize its energy. For flexiblestructures, there may be several conformations of equally low energy as well as the possibility that a conformation other than the minimum energy conformer will participate in the process being modeled. Electronic parameters like HOMO-LUMO gap can vary more among conformations of the same structure than do those for the entire series of chemicals under investigation (17). Thus, when these electronic parameters are used in structureactivity studies, we propose that all reasonable conformations be considered in the analysis (18). Briefly, we previously described a program called 3DGEN, which exhaustively computes all conformations of a given chemical structure (19). The 3DGEN routines use the OASIS program package for QSAR development that has also been previouslydescribed elsewhere(20). The OASIS systems permit the explorationofrelationshipsusing electronic parameters for conformations other than the minimumenergystructas well as usingparametervalues
“+”
TABLE 1
Phototoxicity and HOMO-LUMO Gaps of a=Terthienyl and Toluene Derivatives chemical no. 1 2 3 4 5 6 7 9 10 13 14 15 16 18 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 37
5-substituted -H -Br 5,5”- dibrorno
a-Terthienyl Derivatives darwlight LC50 ratio mosquito
brine shrimp
maximum’
1.45 >3.65
46.6 >66 000 >37 000 250 000 >2 700 51.5 14.6 123 219 2.33 171 403 9.71 31.1 27.6 >10 000 14.2 15.2 7.9 12.7 10.8 14.6 5.3 21 100 19.2 17.3 44.1 11.5 5.9 6.0 54.0
7.50 7.47 7.44 7.34 7.24 7.10 6.90 7.39 7.20 7.38 7.41 7.33 7.43 7.40 7.34 7.30 7.33 7.29 7.24 7.21 7.15 7.24 7.17 7.08 7.33 7.34 7.45 7.32 7.17 6.81 7.85
-I 5,5”- diiodo -SCH3 5,5”-dimethylthio -Si(CH3) -Si (CH3)3-5”-COzH -CHzCHzCOzH -CHzCHzCOzCH3 -CHO -CONH2 -CH2OH -CEN 5,5”-dicyano -CECH -CaCCH3 -CzCCHzCHzOH -CH=CF2 -CHaCC12 -CH=CBrz -CH=CHCOzH -CH=CHCOzCH3 -CH3 5,5”-dimethyl -C(CH3)3 5,5”-d i-t-but y I -NO2 5-H-3”-nitro 5-H-3,4’-diethyl
2.59 > 16.5
4.20 3.66 1.BO >74.5 6.92 > 29.1 5.18
3.21 2.39 >9.41
> 1.07
HOMO-LUMO gap (eV)
.
minimum#
lowest 109’0
lSCF
7.71 7.76 7.68 7.92 7.55 7.30 7.04 7.88 7.42 7.73 7.66 7.49 7.68 7.98 8.01 7.56 7.55 7.69 7.52 7.75 7.33 7.50 7.51 7.66 7.89 7.94 7.70 7.91 7.39 7.91 8.63
7.51 7.47 7.44 7.35 7.24 7.1 1 6.93 7.45 7.26 7.48 7.41 7.39 7.43 7.53 7.35 7.33 7.91 7.32 7.33 7.24 7.19 7.26 7.21 7.22 7.45 7.39 7.52 7.43 7.22 7.65 8.07
8.36 7.72 7.80 7.21 7.31 8.08 7.82 8.19 7.82 8.17 8.18 8.04 8.16 8.08 7.94 8.24 8.13 8.25 8.18 8.17 8.31 7.85 8.17 8.17 8.39 8.14 8.16 8.24 7.07 7.39 8.32
Toluene Oerivatives
HOMO-LUMO gap (eV) no. 1 2 3 4 5 6 7 8 9 10 11 a
substitution 2,4,6-trinitro2,3-dinitro2,4-dinitro2,g-dinitro3,4-dinitro2,3-diarnino2,4-diarnino2,6-diarnino3,4-diarnino2-arnino-4,6-dinitro4-amino-2,6-dinitro-
maximum’
minimum’
lowest 10%
15cf
2
9.36
9.49
9.48
8.77
3 1 1.5 2
9.22 9.25 9.28 9.25 8.61 8.71 8.82 8.54 7.94 7.98
9.32 9.59 9.56 9.50 9.58 9.48 9.65 9.50 8.29 8.88
9.26 9.35 9.48 9.29 8.68 8.72 8.82 8.62 8.05 8.22
8.70 8.78 8.62 8.77 8.45 8.61 8.74 8.33 7.42 7.30
darwlight effect ratio
HOMO-LUMO gap for conformer with maximum and minimum energy, respectively.
that are representative of populations of conformations which meet specific requirements. The ability to specify chemical attributes of the conformations used in this approach offers a highly dynamic method of developing mechanistic QSARs. In our immediate case, the phototoxicity data were too qualitative to try to expect good correlations. At the same time, even the evaluationof the phototoxicity window could be affected by the variation of HOMO-LUMO gap among the many possible conformations of the test chemicals.To accommodate this possible variance, we computed the maximum and minimum values of HOMO-LUMO gap for the conformations as well as the HOMO-LUMO gap of the conformations having energies within the lowest 10 per-
centile, which often was a single minimum energy conformer. These data are reported in Table 1 along with the toxicity data. The most computationally intensive part of this approach is the optimization of molecular geometry in MOPACG needed to calculate the orbital energies,which is a rapid computation. The algorithm 3DGEN provides a reasonable molecular geometry for each conformation, and these estimates are used as a “initial guess” for the more extensive calculations. Using the 3DGEN geometries directly in the calculation of orbital energies via lSCF routines in MOPACG provided a much less time-consuming estimate of HOMO-LUMO gap. Table 1 contains the estimationsof HOMO-LUMOgap of the lowest 10percentile conformers labeled lSCF that were calculated before the computer-intensive geometry optimization process. VOL. 29, NO. 5 , 1995 / E N V I R O N M E N T A L SCIENCE ti TECHNOLOGY
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1269
6.8
7
7.2
7.6 7.8 HOMO-LUMO Energy 7.4
8
8.2
8.4
FIGURE 3. Variation of the darkjlight toxicity ratio for a-terthienyl derivatives with mosquito larvae.
Results and Discussion Figure 1 presents the variation of phototoxicity of PAHs with HOMO-LUMO gap as well as the shaded phototoxicity window fromourprevious work (3). Forthe present study, the calculated dark/light toxicity ratios and the HOMOLUMO gap energies for the a-terthienyl and toluene derivatives are presented in Table 1. For purposes of comparingthese data, we selected the HOMO-LUMOgaps from Table 1for the lowest 10 percentile energy conformations after geometry optimization using the PM3 Hamiltonian in the MOPAC6 package. The dark/light toxicity ratio as a measure of enhanced phototoxicity forthe testswithmosquito larvaevasiedfrom 1.45 in the unsubstituted parent chemical to greater than 74.5. In general, all greater than values were the result of no observed LC50 in the dark test at the highest concentration used. Figure 3 presents the distribution of toxicity ratios along the HOMO-LUMO gap axis. Three of the chemicals tested with the mosquito had less than a factor of 2 increase in toxicity in the presence of light, and all three have HOMO-LUMO gaps greater than 7.5 eV. AU of the other chemicals tested with the mosquito were more phototoxic and have gap energies between 7.1 and 7.5 eV. Clearly, these datasupporttheuseoftheproposedwindow to identify potentially phototoxic chemicals. Chemicals 6 and28 showed only modest enhancement in toxicity, which may be due to decreased hioaccumulation. Although this set of chemicals did not include many examples of those with energy gaps near or less than 7.0 eV, thedatasuggest that thelower limit ofthe phototoxicity window for thiophenes may be shifted to greater energies incomparison to that observedfor PAHs (Figure 1). While such a shift would be reasonable for tests having a higher proportion of W - A or W-B light relative to visible light in comparison to sunlight, the interpretation of the data as that level of detail is confounded by the fact that Marles et al. (14) did not normalize the toxicity measurements for differential bioconcentration. For example, chemicals numbered 10 and 28 are carboxyl derivatives for which the bioconcentration would he expected to he several orders of magnitude less than other derivatives. The lack of enhanced toxicity in the presence of light should be highly attenuated by the fact that the chemical does not accumulate as a residue in the larvae. 1270. ENVIRONMENTAL SCIENCE &TECHNOLOGY IVOL. 29. NO. 5.1995
The toxicity data obtained from brine shrimp included manymorethiophenes coveringabroaderrangeofHOMOLUMO gap (Figure 4). Again, the highest d u e s of dark/ light ratios for toxicity were from those SUUCNreSforwhich the energy gap was between 7.1 and 7.5 eV. Halogenated and cyano derivatives tended to be much more toxic than the others while the introduction ofwater-soluble moieties decreased the phototoxicity, even within the phototoxicity window. Chemicals such as numbers 13, 16,24,34,and 28allhavepolarsustituentsthatshoulddramaticallyreduce hioconcentration. The m e relative phototoxicityofthese chemicals cannot be determined without tests which allow the dark/light toxicity ratio to he corrected or at least normalized for residue concentration in the organism. Indeed, Marles et al. (14) discussed the importance of hydrophobicityin the results they presented and attempted to fit the data to a parabolicmodelwithrespectto the octanol/waterpartition coefficient. Such models are bound to fail because they assume constant photoactivation for all chemicals. Our analysis suggests that hydrophobicityand HOMO-LUMO gap both control the experimental phototoxicity. The influence of the latter can only he reliably examined if bioaccumulationisheld constant as Newsted and Giesy (1) did with PAHs. If a multivariate model is to be developed, chemical selection and test conditions would have to be much more carefully controlled. These data strengthen the idea that the HOMO-LUMO gap is a good discriminator of phototoxic chemicals. Consistent results have been attained with three species of aquatic organisms and with both rigid and flexible delocalized chemicals. Table 1 shows that modem computational methods for the HOMO-LUMO gap can produce variable estimates that vary by almost 1eV, depending on the conformation of the chemical selected for modeling. We suggest that using the average HOMO-LUMO gap for the conformationsin the lowest 10 percentile with respect to total energy might he a reasonable approach. Table 1 also shows clearly that the unoptimized structures yield HOMO-LUMOgap energies whichvarywidelywithrespect to those from optimized structures. The value for the unoptimized 5-nitro derivative was below the entire range of optimized d u e s , while the values for the unoptimized alkyl derivatives were generally greater than the optimized
Q
.E 5
3.5
a
3
G
2.5
E
2
9
1.5
.-c
0 ._
20
1
37
3
2m
e. 0 9 0 -
0.5
0 6.0
7
7.2
7.4 7.6 7.0 HOMO-LUMO Energy
0
0.2
0.4
FIGURE 4. Variation of the darklligM toxicily ratio for a-terthienyl derivatives with brine shrimp.
maximum. The inconsistency of these results indicate that computing shortcuts such as using unoptimized structures are not reliable enough, even in the initial screening of chemicals. The phototoxicity data for amino- and nitrotoluene derivatives presented in Table 1 provide an interesting evaluationof our model. All of the chemicals have HOMOLUMO gaps greater than 7.94 eV regardless of the computationmethod, whichiswelloutside theupper boundary of the phototoxicitywindow proposed previously. These chemicals would he classified as non-phototoxic based on the mechanism underlying the model. The data show that, in fact, most of the toluenes tested were not significantly phototoxicinthe testswithD. magna. However, Davenport et al. (15) found that light did increase the developmental effects of the toluidines and trinitrotoluene in sea urchins. The photoinduced effects are especially noteworthy because these toluene derivativeswould all be expected to have very low bioaccumulation potentials because of the polar substituents. If the chemicals were not expected to accumulate as cellular residues nor to produce oxygen radicals through photoinduced excited states because of the large HOMO-LUMO gap, the apparent phototoxicity merits further study. Strict interpretation of these results is hampered by the test methods used by Davenport et al. (1% as well as the fact that these chemicals produced toxic effects in the dark and the light. It is plausible that light is influencing the sea urchin development through a different mechanism. Davenport et al. (15)found that none of the amino- and nitrotoluene derivativesproduced phototoxicity in E. coli or D. magna. This is consistent with our model. These derivativesalso have unique redox properties and may be involved in equilibria with common electrophilic intermediates. N-Oxide or hydroxide intermediates may be a better explanation for the abnormal development of sea urchin embryos because the early embryogenesis is particularlysensitive to electrophiles (21).Theenhancingeffect ofW-Alight ononlythedevelopmentalendpointssuggests that light stimulated greater embryo metabolic activation of the toluidines or that these chemicals can form small amounts of other radicals for which the embryo test is especially sensitive. Because of the relevance of these
questions to the potential hazards of munitions wastes, more detailed studies of the effects observed by Davenport et al. (15) are needed.
Conclusions This study evaluated a model of photoinduced toxicity in aquatic organisms that used the computedHOM0-LUMO gap energyofthe ground-state molecule. The phototoxicity window developed for PAHs, where HOMO-LUMO gap energy ranged from 6.7 to 7.5 eV, was compared to the evidence of phototoxicity for a series of a-terthienyl and toluene derivatives. Despite the differences in the light source and the absence of normalization for hioconcentration. the data on a-terthienyl pesticides agreed well with the proposed model. Those chemicals showing the greatest increase in toxicity in the presence of light had HOMOLUMO gaps in the range of 7.1-7.5 eV. The amino- and nitrotoluenes all have HOMO-LUMO energies greater than 8 eV, which lies outside the original phototoxicitywindow. Five of the 11 toluenes were not phototoxic; however, the diaminotoluenes with HOMO-LUMO gap from 8.6 to 8.8 eVappearedtocausegreatereffectsinlightthanindarkness in one of three assay systems. The possibility of other biochemical and photochemical mechanisms being involved in the toxicity of these chemicals should he studied.
Acknowledgments The authors thank Dr. Steven Bradhury and Ms. Christine Russom of the EnvironmentalResearch Laboratory-Duluth for their perspective in analyzing the phototoxicityexperiments. This work was supported by US. EPACooperative Agreement CR820433. Mention of software or computer systems does not imply endorsement by EPA or the University of Wisconsin-Superior.
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(6) Kagan,J.; Taderna-Wieland,K.; Chan, G.; Dhawan, S. N.;Jaworsky, J.; Prakash, I.; Arora, S. K. Photochem. Photobiol. 1984,39,465467. (7) Gant, T. W.; Rarnakrishna, R.; Mason, R. P.; Cohen, G. M. Chem.Biol. Interact. 1988, 65, 157-173. (8) Cornporti, M. Chem.-Biol. Interact. 1989, 72, 1-56. (9) Pechrnann, J. H. K.; Wilbur, H. M. Herpetologica 1994, 50, 6584. (10) Yoon, C. K . N. Y. Times 1994, Mar 1 . (11) Veith, G. D.; DeFoe, D. L.; Bergstedt, B. V.J. Fish. Res. Boardcan. 1979, 36, 1040-1048. (12) Clernents, R. G.; Boethling, R. S.; Zeernan, M.; Auer, C. M. In
Environmental Risk Assessment for Organochlorine Chemicals; SETAC Foundation Special Symposium; Nottawasaga Inn: Toronto, Canada, 1994. (13) Ghquez, J. L.; Martinez, A.; MBndez, F.J. Phys. Chem. 1993,97, 4059-4063. (14) Marles, R, J.; Cornpadre, R. L.; Cornpadre, C. M.; Soucy-Breau, C.; Redmond, R. W.; Duval, F.; Mehta, B.; Morand, P.; Scaiano, J. C.; Amason, J. T. Pestic. Biochem. Physiol. 1994, 41, 89-100. (15) Davenport, R.; Johnson, L. R.; Schaeffer, D. J,; Balbach, H. Ecotoxicol. Enuiron. Sat 1994, 27, 1099-1112.
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(16) Stewart, J. J. P. MOPACG; Frank J. Seiler Research Laboratory, U.S.Air Force Academy: Denver, CO, 1990. (17) de Cornpadre, L.; Debnath, L. R.; Shuster, A. J.; Hansch, C. Environ. Mol. Mutagen. 1990, 44-55. (18) Mekenyan, 0. G.; Ivanov, J. M.; Veith, G. D.; Bradbury, S. P. Quant. Struct.-Act. Relat. 1994, 13, 302-307. (19) Ivanov, J. M.; Karabunarliev, S. H.; Mekenyan, 0.G.J. Chem. InJ Comput. Sci. 1994, 34, 234-243. (20) Mekenyan, 0. G.; Karabunarliev, S. H.; Bonchev, D. I. Math. Chem. 1990, 4, 207-215. (21) Schultz, T. W.; Ranney, T. S.; Riggin, G. W.; Cajina-Quezada, M. Trans. Ani. Microsc. SOC. 1988, 107, 113-126.
Received for review August 3, 1994.Revised manuscript received January 3, 1995. Accepted January 18, 1995.@ ES940491L @Abstractpublished in Advance ACS Abstracts, March 1, 1995.
