Environ. Sci. Technoi. 1993, 27, 2795-2800
A Radioactive Tracer Technique for the Study of Lethal Body Burdens of Narcotic Organic Chemicals in Daphnia magna Andrew V. Pawlisz' and Robert H. Peters
Biology Department, McGill University, 1205 Dr. Penfield Avenue, MontrQal,Quebec, H3A 1B1 Canada High analytical costs and high detection limits have constrained experimental analyses in aquatic ecotoxicology. To circumvent these restrictions, we have assessed a novel radiotracer approach to tissue residue analysis, using 10, I4C-labelednarcotic organic chemicals. Daphnia magna was exposed to potentially lethal concentrations of acetone, acetophenone, benzene, benzoic acid, butanol, 1,2-dichlorobenzene, 2-methylnaphthalene, 1,1,2,2-tetrachloroethane, 1,2,4-trichlorobenzene, and toluene. Analysis of the body burdens after 48-h exposure showed that the radiotracer method could measure chemical levels in single Daphnia at body concentrations ranging from 0.02 to 6310 mmol/kg, depending on the chemical. The lowest body residue of 0.02 mmol/kg was well above the lower limits of detection of =lnmol/kg. The technique measured body residues of lipophobic (Kow= 0.55, acetone) and lipophilic (Kow= 16 000,1,2,4-trichlorobenzene)chemicals equally well. The radioactive tracer technique is simple, sensitive, widely applicable, and economical.
Table I. Literature Estimates of Molecular Weights, Solubilities, and Partition Coefficients (ICow) with Standard Deviations of 10 Narcotic Organic Chemicals. chemical compound
log molecular solubility weight (mmol/L)
infinite
acetone
-0.26 f 0.03 7 (8,14,19-22) 3.01 0.88 (8,14,19) 4 (20) 1.69 1.6 f 0.05 (23) 3 (8,20,23) 1.35 1.9 f 0.08 (14) 4 (26,27, 32,33) 2.1 f 0.01 1.36 (18,21) 15 (4,5,20-31) 0.75 2.6 f 0.06 (21,23,29) 3 (20-22) 2.76 2.8 0.3 (21) 6 (8,14,20,21,35) -0.18 3.4 f 0.8 (21) 3 (5,20,21) -0.75 3.9 (34) 2 (20) -0.72 4.2 f 0.3 (21) 8 (14,20,21,23,25,36) (18)
butanol acetophenone
120 (18)
benzoic acid
122 (18)
benzene toluene 1,1,2,2-tetrachloroethane
167 (18)
Introduction
(&%%N
1,2-dichlorobenzene
147 (18)
*
The standard bioassay is the most common method of toxicity assessment for chemicals. In this test, organisms are exposed to several concentrations of pollutant under otherwise constant environmental conditions to determine the concentration needed to affect half the population (1-3). Although this approach may be adequate for testing a few chemicals, it is too costly and time-consuming for the thousands of chemicals already in production and the hundreds more invented every year (4). The cost of the bioassays stems from the need for many replicates at different concentrations, requiring many organisms and much preparation. The standard tests are also sensitive to factors like body size and time of exposure that affect replicability of results and must therefore be thoroughly controlled (5,6). Finally, standard assays cannot address the toxicological processes taking place inside animals because internal levels are unknown. A bioassay using the internal concentration of pollutants would reduce many of these problems. However, chemical analysis of contaminants in individual animals using available methods would be even more costly, timeconsuming, and, for small animals, technically difficult. An alternative method is needed. A radioactive tracer technique might be such an alternative. In this method, a chemical compound is detected by the radioactivity of the attached isotope. Although radiotracers are widely used in studies of bioconcentration ( l ) they , have largely been overlooked in other types of ecotoxicological investigations. The object of the current work is to examine aradioactive tracer technique for assessment of the body residues of narcotics in a single test species, Daphnia magna, in terms of sensitivity, replicability, economy, and applicability. Ideally, the technique should be sufficiently sensitive to
Narcotic Organic Chemicals. Ten narcotic organic chemicals were selected because they are commercially available in a 14C-labeledform, identified as narcotics (7, 8),well-described toxicologically in the literature, important as prioritypollutants (9),and representative of a wide range of physicochemical properties, chemical structures, and acute toxicities (Table I). Before use, the radiolabeled chemicals were diluted with the nonradioactive carrier to increase the amount of
@ 1993 American Chemical Society
Environ. Scl. Technol., Vol. 27, No. 13, 1993 2795
0013-936X/93/0927-2795$04.00/0
2-methylnaphthalene
142 (18)
1,2,4-trichlorobenzene
181 (18)
Cited references are listed in parentheses.
detect minute quantities of different chemicals inside single D. magna and, thus, to allow assessment of interindividual differences. Furthermore, the data obtained by this method should be no more variable than those in standard assays. The technique should also be precise enough to distinguish body concentrations of D. magna that were dead, immobilized, or apparently unaffected by the exposure to pollutant, because the ability to detect differences in the chemical body residues between affected and "unaffected" animals would help to quantify the internal levels needed to kill or to immobilize D. magna. Finally the technique should be economical enough to allow cost-effective data collection.
Experimental Section
Table 11. Source, Purity, Supplied and Adjusted Specific Activities, and Minimum Detectable Levels of 10 Narcotic Organic Chemicals Used in Current Studys min specific activity detectable purity supplied adjusted level (% ) (GBqlmmol) (MBq/mmoI) (pmollkg) chemical source AC BUT ACP BA BEN TOL TCE DCB 2MN TCB
Sigma Sigma Sigma Sigma Sigma CHEMSYN CHEMSYN Sigma Sigma Sigma
>98 >98 >98 >98 >98 >98 >86 >98 >98 >98
0.10 0.29 0.65 0.08 1.87 0.08 0.18 0.33 0.33 0.20
0.27 0.014 1.24 5.6 1867 13.3 13.8 0.28 325 0.27
1.57 0.03 0.35 0.08 2.30 X 0.03 0.03 1.54 1.32 X 1.58
0.01 l@3
Table 111. Rearing and Test Conditions of Daphnia magna variable
condition
light photoperiod (h) temperature (“C) culture vessels no. of animals per vessel feeding frequency feeding amount
3X Sylvania Cool White (20 W)
16 light/8 dark 20 f 1 1000-mL glass beakers 75 daily 100 mL of “Green Water”
compound available and to reduce experimental costs. Unlabeled forms of the 10 chemicals were obtained from Sigma Chemical Co. (St.Louis, MO) or Beckman Chemicals (Mississauga, ON). Their purities, as determined by the manufacturer, were above 98% in all cases, except for 1,1,2,2-tetrachloroethanewhose purity was greater than 86% (Table 11). Enough of each unlabeled compound was mixed with its corresponding labeled form to increase the amount of the radiolabeled compound while maintaining specific activities sufficiently high that radioactivities of individual animals were at least 1 order of magnitude above background. Dilution increased the amount of radiolabeled compound available for experimentation by as much as 1000 times. Test Organism. D. magna was selected because it is well-known toxicologically, because it is easily kept, and because cladocerans represent a major element of aquatic food webs. The clear carapace of D. magna allows easy inspection of the activity of the heart, gut, and filtering apparatus and, therefore, simplifies the determination of whether the animal is alive or dead. The original stock of D. magna was obtained from Environmental Biology Annex I, University of Guelph, Ontario. Parent cultures were kept in 1000-mL glass beakers containing standard reconstituted water ( I ) and about 75 individuals per beaker. To assure that the Daphnia were well-fed, the cultures were given enough algal suspension from a goldfish tank to keep the culture water slightly green (10). All beakers were kept in an incubator (photoperiod = 16-hlight, 8-h dark; temperature = 20 “C), and the culture vessels were cleaned and water was replaced weekly. Daphnia culture conditions are summarized in Table 111. Envlron. Scl. Technol., Vol. 27, No. 13, 1993
50
le
The activity was adjusted by dilution with the unlabeled forms of these chemicals. The chemical codes are acetone (AC), acetophenone (ACP), benzene (BEN), benzoic acid (BA), butanol (BUT), 1,2-dichlorobenzene (DCB), 2-methylnaphthalene (2MN), toluene (TOL), 1,1,2,2-tetrachloroethane(TCE), and 1,2,4-trichlorobenzene (TCB).
2796
100
0.1 1 Body Burden (mmol/kg)
10
Figure 1. Cumulative percent distributions of the body burdens of 1,Pdichlorobenzenein dead (d), immobilized (I), and unaffected (u) 0. magna after 48-h exposure to the potentlally lethal concentrations(EC = 48-h LCs0) of these compounds. The number of observations composing each curve are given in the corner of the panel. The exposure concentrations are based on 48-h LC50 reported in the superscripted references in parentheses. Dash-dot line indicates the 10 % cutoff.
Tests Using Radiolabeled Compounds. Test solutions of the radiolabeled compounds were prepared by placing an aliquot of each chemical into a volumetric flask containing standard reconstituted water (1). The concentration of each solution was based on Daphnia 48-h LC~O’S of each chemical reported in published sources (Figures 1and 2). The flask was then sealed with arubber stopper and wrapped in aluminum foil. The flask was shaken gently by hand for few minutes and then placed on a magnetic heater-stirrer at 23 OC. Enough time wao allowed (24-72 h) for the chemicals either to diasolve completely or to form a saturated solution. Water concentrations of the narcotic compounds were measured after the preparation of the test solutions and after each experimental run. The loss of contaminant during the run was about 10%. The measurement consisted of placing 50-pL aliquots of test solution in scintillation vials with 2.0 mL of Ready Protein (Beckman, Toronto, Ontario) scintillation fluid. Vials were then vortexed, and their radioactivities were determined with a Wallac LKB (Turku, Finland) scintillation counter. Later, the radioactivities were converted into chemical concentrations and expressed as millimole per liter. Each experimental run began when about 15acclimated, parthenogenic females of different sizes were placed into each of eight, 20-mL, screw-top vials containing 18 mL of test solution. Two other vials contained no narcotic and served as controls. Each run was replicated 4-7 times. No food or replacement water was provided during the tests. After 48 h, “swimmingnand “nonswimmingndaphnids were collected separately with a large-bore pipet. Each group was deposited onto a small tray made of Nitex screen and rinsed with 10 mL of clean water to reduce contamination by external chemicals. The tray containing nonswimming Daphnia was placed under a dissecting microscope, and the animals were examined for life signs. The absence of movement of the appendages, digestive tract, and heart indicated death. The presence of any of these life signs indicated narcosis. Body lengths of all in millimeter as the distance between the eye animals (L, and the base of the tail spine) were measured using an ocular micrometer. These were later converted to body mass ( W ,in microgram of fresh weight) using a relationship developed as part of the current study (log W = log 1.98
100 50
100 50
100 50
0.01
0.1
1
10
100 0.1
1
10
100 1
10
100 100010000
Body Burden (mmol/kg) Flgure 2. Cumulative percent distributions of the body burdens of nine narcotic organic chemicals in dead (d), immobilized (i), and unaffected (u) D. magna after 48-h exposure to the potentially lethal concentration (EC = 48-h LCE0)of these compounds. The number of observations composing each curve are given in the corner of the panels. The exposure concentrations are based on the 48-h LCsoreported within superscripted references in parentheses.
+ 3.39 log L;r = 0.93; P < 0.0001; n = 94). Sorting, rinsing,
and measuring were done as quickly as possible to reduce the possible loss of radiochemicals through evaporation. Internal Residue Analysis. Once each organism was sized and its life signs had been examined, it was placed individually in a 7.0-mL glass scintillation vial containing 2.0 mL of Ready Protein. The tubes were left undisturbed overnight in a cool room to allow for digestion of tissue and subsidence of chemiluminescence. Next, each vial was vortexed for 15 s and analyzed for radioactivity. The counting time was adjusted so the counting error was less than 2 % of the mean. After correction for background radiation, the measured radioactivities were expressed as millimole of contaminant per kilogram of wet body weight. The calculations of the lower detection limits (Table 11) of the narcotic organic chemicals in D. magna were based on values of the minimum detectable radioactivities. These minima were calculated as LLD,, = 4.669, (1) where LLD96 represents the lower limit of detection of radioactivity at the 95% level of confidence, and s b represents the standard deviation of the background counting rate ( I ) , 12.3 cpm. Statistical Analysis. All statistical analyses used SAS analytical software (11). Body burdens were log transformed to stabilize variance (12), and the individual estimates of the body burdens of D. magna for each
experimental vial were combined into means to reduce pseudoreplication (13). A nested ANOVA was performed to determine what proportion of the variance in Daphnia body residues of narcotic chemicals could be associated with differences among the chemicals, physiological responses within chemicals, among experimental vessels within physiological response groups and chemicals, and among replicates within vessels, responses, and chemicals. Tukey’s test (14)was used to determine whether significant differences exist among the mean body residues and among the standard deviations of these mean body residues of the unaffected, immobilized, and dead animals (Le., the physiological response groups).
Results and Discussion Analysis of Technique. The results for a typical chemical, 1,2-dichlorobenzene, are presented as the cumulative percent distributions of body burdens of this compound in unaffected, immobilized, and dead D. magna after 48-h exposure (Figure 1). The greatest body burdens of this compound (80 mmol/kg) were found in dead animals. The next highest maximum occurred in immobilized Daphnia (50 mmol/kg). The smallest maximum was found in unaffected organisms (10 mmol/kg). At the other extreme, the minimum quantities of 1,2-dichlorobenzene taken up were more similar, but the unaffected animals had the least (0.3 mmol/kg), the immobilized had Environ. Sci. Technol., Vol. 27, No. 13, 1993 2797
the highest minimum (0.5 mmol/kg), and dead animals were intermediate (0.4 mmol/kg). Because extreme values of the body residues are highly variable and likely depend on sample size, we have elected to omit the 10% most radioactive and the 10% least radioactive animals for each chemical and physiological response group. Omitting the extreme values resulted in a notablysmaller range of the body residues. For instance, in the case of 1,Bdichlorobenzene (Figure l),the range in logarithm of the body residues shrinks by about 2 orders of magnitude from 1000 to 10 for each of the unaffected, immobilized, and dead Daphnia. Similar reductions are achieved for the other nine chemicals (Figure 2). Considerable variability of the body burdens remains. Figures 1and 2 indicate that the dead, immobilized, and unaffected Daphnia had similar ranges of internal body residues and that the cumulative curves crossed several times between 0.5 and 8 mmol/kg. Overlapping of the cumulative curves indicates different tolerances to 1,2dichlorobenzene among individual Daphnia. Thus, at any given burden, some animals are killed, whereas others are only immobilized, and still others appear unaffected. Similarly, the contaminant burdens of animals sharing similar responses may differ as much as 100 times, if extreme values are considered. Similar patterns occur for each of the other nine chemicals (Figure 2). Mean body burdens of the unaffected, immobilized, and dead Daphnia differed significantly, but the cumulative curves overlapped. Consequently, animals with similar responses often had different body burdens, and animals with the same body burdens often differed in response. This is further indicated by the variation in burden among animals that were similarly affected, as represented by the span of curves for different responses as in Figures 1and 2. Such variation in response is troublesome because a given internal concentration is associated with several responses. As a result, prediction of Daphnia responses from the internal levels of toxicants is necessarily probabilistic. Quantities of narcotics detected ranged from as low as 0.02 mmol/kg (2-methylnaphthalene) to as high as 6310 mmol/kg (butanol). The ability of the radioactive tracer technique to detect such a wide range of narcotic body burdens in single Daphnia is notable. We had initially supposed that several Daphnia would be needed to obtain enough radioactivity to estimate body burden estimate, but one can consistently obtain body residue estimates for single organisms. For instance, the minimum quantity of benzene detected in single Daphnia was about 9 nmol. However, much lower values could have been detected, because this amount was 5 orders of magnitude above the detection limit of 520 pmol/animal for this chemical. The cases for the other chemicals were similar, for the internal concentrations (Figure 2) were above the lower detection limits of the technique (Table 11). We had also feared that the radioactive tracer technique would not be able to detect measurable quantities of chemicals with low octanol-water partition coefficients (KO,), because such compounds are not readily accumulated by organisms (15). However,the experiments showed that both lipophobic (acetone; KO,= 0.55) and lipophilic (1,2,4-trichlorobenzene; KO,= 16 000) compounds were detectable in individual organisms (Figure 2). Thus, the radioactive tracer technique applies to chemical compounds with very different properties. 2798
Envlron. Sci. Technol., Vol. 27, No. 13, 1993
Table IV. Log Mean Body Burdens and Standard Deviations of 10 Narcotic Compounds in Unaffected, Dead, and Immobilized Daphnia magna after 48-h Exposure to of These Potentially Lethal Concentrations (484 LC~O) Compoundsa chemical AC BUT ACP BA BEN TOL TCE DCB MEN TCB means
mean body burden (SD) unaffected dead immobilized mean SD 1.458 (0.35) 1.75 (0.44) 1.548 (0.34) 1.83 (0.29) 2.55 (0,301 2.30 (0.35) 0.27 (0.25) 0.81b (0.28) 0.83b (0.30) 0.43 (0.67) 0.32 (0.53) -0.37' (0.71) -4.52' (0.83) 0.17' (0.52) -0.01 (0.60) 0.10d (0.64) 0.26d (0.39) 0.02s (0.47) 0.36e (0.48) 0.4~5~ (0.47) -0.93 (0.53) -Q.37f(0.45) -0.42f (0.38) -1.909 (0.45) -0.95gh(0.49) -0.84h (0.54) 0.65 (0.063) 1.32 (0.55) 1.10 (0.30) 0.50 (0.39) 0.14 (0.44) 0.53 (0.50)
0.38 0.31 0.28 0.57 0.54 0.47 0.45 0.49 0.30
Chemical codes and the number of estimates composing each mean are given in Table I1 and Figures 1and 2. Standard deviations are not significantly different from each other at the 5% level. Identical superscripted letters indicate that differences in the means within chemicals are not significant (P> 0.05; Tukey). All other numbers differ significantly (P< 5%). -
-
~
_
l
s
l
_
Table V. Summary of Nested ANOVA Performed on Tissue Residues of Narcotic Compounds in Daphnia magnae source
DF
sum of squares
mean square
Fvalue
model error corrected total chemical state between vials within vials
611 3661 4272 9 23 121 458
4903.30 419.90 5323.10 4494.90 118.70 148.50 141.20
8.00 0.10
69.97
0.00
499.40 5.20 1.20 0.30
4354.60 45.01 10.70 2.69
0.00 0.00 0.00 0.00
PR> F
The variance was partitioned among the chemical compounds (chemical), response of the organism within chemicals (state), replication within states and chemicals (replication), and vials containinganimalswithin replicates, states, and chemicals (vial) (root MSE = 0.30; R2 = 0.90; and CV = 43.9).
The 14C-labeledtechnique might appear to be unattractive due to the expense of the radioactively labeled chemicals: a minimum order cost at least $250 (U.S.). However, because each test required so little radiolabeled chemical, the 14C-labeled technique proved relatively economical. A single test, consisting of 50 vials each containing 15 individuals, cost roughly $10. The estimated cost of a residue estimate of narcotic compounds for individuai Daphnia was therefore about $0.10, Hence, despite the high cost of the radiolabeled compounds, the radioactive tracer technique is economical enough to permit collection of much data. The replicability of results can be assessed as the standard deviations of the logarithms of body burden estimates, calculated for each response and chemical. These standard deviations ranged from 0.063 for 1,2,4trichlorobenzene to 0.83 for butanol (Table IV), but Tukey's comparison (13)revealed that the mean standard deviations were not significantlydifferent among chemicals ( P > 5%). Variability in the body residue estimate was therefore similar for all chemicals and responses. Differences in the mean burdens of the different chemicals were identified by nested ANOVA (Table V). The mean body residues of the 10 narcotic compounds
were significantly different at the 5 % level,and differences among chemicals explained 84% of the variance in' the body residues. Differences among the unaffected, immobilized, and dead Daphnia explained only 2.2% of the total variance, but these differences were still significant (F-test). Differences in the experimental vessels within and among replicates contributed only 2.7% and 2.8% of the total variance in the body residues of narcotics, respectively, and again the F statistics showed these differences to be significant at the 5 % level. In other words,the body residues were most affected by differences in the chemical compounds and not by differences in experimental vessels, experimental runs, or Daphnia tolerances. Therefore, improvements in the experimental protocol could only reduce the overall variability in the body residues by 16% at most. Possible sources of the variability attributable to methods include the variable efficiency of rinsing of the animals. However, this influence was small as the analysis of few exuviae shed during experiments indicated that the amount adsorbed to Daphnia surfaces was only 0.014% of the amount found in the tissues. Mean Body Burden and Response. The nested ANOVA revealed significant differences in mean body residues among the unaffected, immobilized, and dead animals. These differences ranged from 0.5 (1,2-dichlorobenzene) to 0.8 (acetophenone) orders of magnitude (Figures 1and 2). Because ANOVA also showed that these differences in animal responses contributed only 2.2% of the total variance, Daphnia could be analyzed irrespective of their response. However, since there are significant differences among the three groups, precision may be improved if one separates the unaffected, immobilized, and dead Daphnia. Mean body burdens of narcotics of unaffected Daphnia (1.1mmol/kg; antilog 0.041) were 2-3 times lower than the mean body burdens of the immobilized (2.5 mmol/kg; antilog 0.40) or dead Daphnia (2.7 mmol/kg; antilog 0.43; Table IV). For the individual chemicals, the unaffected individuals exhibited lower body burdens of narcotics than immobilized or dead animals in all cases except benzoic acid for which the unaffected Daphnia had significantly greater body burdens than the affected animals. These differences were consistently significant at P < 0.05 (Tukey), except in the case of acetone, benzene, 1,1,2,2tetrachloroethane, and 2-methylnaphthalene (Table IV). No consistent differences were found in the mean body burdens of narcotic compounds of the immobilized and dead animals. In fact, the difference between the mean body residues of the dead and immobilized animals was no more than 1.0 mmol/kg. Apparently, either our separation of dead from immobilized animals was imperfect or immobility so slows uptake that, once narcotized, animals die before accumulating much more of the narcotic. In either case, the results imply that immobilized and dead daphnids could be combined without altering the result. This has the advantage of eliminating the time-consuming microscopic examination of vital signs. For all chemicals, similar exposures result in quite different body burdens, and similar body burdens may result in different responses. Differences in burden after 48-h exposure indicate that net uptake rates vary among animals, but we cannot distinguish whether this reflects differences in chemical uptake, in loss, or in both. The response a t a given burden is also variable, but our
experiments cannot show what permits or causes this variability in the response to these body burdens in Daphnia. Possible explanations include differences in the sensitivities of individual D. magna (15) that reflect differences in the lipid content, in detoxification mechanisms, or in the rate of net uptake and depuration of these organisms (16,17). Drawbacks of the Method. The 14C-labeledtechnique has some drawbacks. The major drawback of the technique is that one measures l4C rather than the pollutant. This can result in an overestimation of body residues of contaminants that are metabolized or detoxified by animals. In that case, the parent compound is degraded into harmless metabolites which accumulate in the tissues and so inflate the estimated body burden. Because the technique cannot distinguish between metabolites and the parent compound, we were unable to decide whether readily metabolized compounds such as acetone, butanol, and 2-methylnaphthalene were in fact degraded by Daphnia, thus contributing to their observed high body burden. The technique also depends on the availability of the radiochemical. Only a small number of the common pollutants are readily available in the 14C-labeledforms. These compounds are expensive, and custom-made radiolabeled pollutants are even more costly. Furthermore, because a radioactive isotope is used, there is a need for licensed operators, designated experimental space, a scintillation counter, and special facilities for handling and disposal of waste.
Conclusions Despite its drawbacks, the radioactive tracer technique is an attractive method for the determination of contaminant tissue residues. The radioactive tracer technique offers several advantages for the measurement of the body concentrations of contaminants. Radioactively labeled compounds can be detected in such minute quantities that their levels in single D. magna can be measured, permitting the calculation of inter-individual variability and the quick and easy collection of large samples. The radioactive tracer technique can handle chemicals of different physicochemical properties, so that all compounds, narcotic or not, can be analyzed similarly. A further advantage of this test is the high probability of detection of the original compound as long as the material is supplied in a pure form, and the metabolic breakdown is slow. Moreover, since comparatively little of the compound is needed, only small volumes of the toxic compounds have to be handled or discarded. The technique is also inexpensive. Drawbacks of the 14C-labeledtechnique include inadequate radiochemical availability, high cost of radiochemicals,need for licensed operators, special handling facilities, major equipment, radioactive waste disposal, and possible confusion of the pollutant and its breakdown products. Despite these negative features, the radioactive tracer technique can be used successfully in the measurement of the body concentrations of various chemical compounds, and the method represents a useful additional tool for ecotoxicological assessment. Literature Cited (1) APHA. Standard Methods for the Examination of Water and Wastewater, 17th ed.; American Public Health Association: Washington, 1989. Environ. Scl. Technol., Vol. 27,
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Received for review March 1, 1993. Revised manuscript received July 20, 1993. Accepted July 30, 1993." Abstract published in Advance ACS Abstracts, November 15, 1993.
