Environ. Sci. Techno/. 1995, 29, 664-672
Isomer and Enantioselective Degradation of Hexachlomcyclohexme Isomers in Sewage Skdye under Anaerobic Conditions HANS-RUDOLF B U S E R * A N D MARKUS D. MULLER Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland
The degradation of the four most common hexachlorocyclohexane (HCH) isomers in sewage sludge was studied using chiral high-resolution gas chromatography/mass spectrometry. Pure isomers and the technical HCH mixture were incubated with sludge from a communal sewage sludge treatment plant. High enantioselectivity was observed for a-HCH with the (+)-enantiomer faster degraded than the (-)-enantiomer. The degradation rates of the different HCHs were in the order of y-HCH > a-HCH > 8-HCH > P-HCH with half-lives between 20 and 178 h for y - and P-HCH, respectively. The rates correlate with the number of axial Cl's in an isomer. Degradation in active sewage sludge was predominantly biotic (80-95%), as compared to the slower degradation in sterilized sludge. However, degradation in sterilized sludge was still significantlyfaster than hydrolysis in water. This enhanced chemical degradation must be due to additional compounds present in sludge and may possibly involve surfacecatalyzed reactions. Suspected initial metabolites of HCHs such as tetra- and pentachlorocyclohexenes (TCCHs, PCCHs) and -hexanes were not detectable, presumably because these compounds degrade even faster than they are formed. Despite the faster degradation of (+)-a-HCH under anaerobic conditions and its faster degradation in surface waters (North Sea, Baltic Sea, Canadian freshwater lakes) under aerobic conditions, (+)-a-HCH is more accumulating in most aquatic biota. General conclusions on the fate of chiral compounds in the environment and on consequences for the monitoring of such compounds using enantio- and nonenantioselective analyses are given.
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Introduction Hexachlorocyclohexane (HCH) was among the most important insecticides for use in agriculture, in forestry, and as a wood preservative ( 1 ) . Cumulative world production likely has reached severalmillion tons (2). HCH is produced by chlorination of benzene under UV light leading to a mixture of various isomers (3). Typically, the technical mixture consists of 60-70% a-HCH, 5-12% P-HCH, 1015% y-HCH, and 6-10% 8-HCH and smaller amounts of other isomers and congeners. y-HCH (lindane) is reported to be the only isomer with insecticidal properties and is isolated from the technical mixture by crystallization (3). Nowadays,the use of technical HCH has been discontinued in most industrialized countries (North America, Western Europe and Japan) but likely continues in Third World countries (4). The physicochemical properties, the levels, and the fate of HCHs in the environment have been extensivelyreviewed (5, 6). Briefly, HCHs are more volatile than many other chlorinated pesticides; they are reasonably water soluble but accumulate to some degree in sediments. Amajor route of dissipation from treated areas is byevaporation followed by aerial transport. Nowadays, still considerable levels are observed in air, rain, and surface waters (6),generally with concentrations of a-HCH > y-HCH pointing to recent uses of the technical mixture. Additionally, the photochemical and the anaerobic microbial transformation of y-HCH into a-HCH have been reported as possible routes to account for increased levels of a-HCH (7, 8). P-HCH seems metabolically most resistant and is generally the most abundant isomer in mammalian species (9). Despite the extensive data available on HCHs, the fate of these compounds in the environment is still not fully understood. In particular, the distinction between biotic and abiotic processes is difficult. As the only HCH isomer, a-HCH is chiral and thus exists in two enantiomeric forms (optical isomers). Enantiomers may show different behavior in biotic processes (chiral environment) whereas their behavior is identical in abiotic processes. Enantioselective analyses may thus allow a differentiation between enantioselective biotic (microbial, enzymatic) from nonenantioselective abiotic processes (chemical, photochemical, distribution, transport). Nevertheless, the chiral aspects of a-HCH and HCH metabolites have so far received little attention (10, 11). The occurrence of HCHs and other chlorinated micropollutants in waste water treatment plants has been reported, but their fate during this treatment process is still poorly documented (12,131. Potential removal processes during this treatment are biodegradation, chemical degradation, adsorption to particulates, and air stripping. Adsorptive compounds such as the HCHs and others are retained with the sludges formed during this treatment, and these sludges need further treatment before they can be used as fertilizer in agriculture. In this study, we describe the behavior of the four most common HCH isomers in sewage sludge from the anaerobic digestor of a typical Swiss waste water treatment plant. Significant degradation was observed for y - and a-HCH. High enantioselectivity in the degradation of a-HCH was indicated by largely different rates for the (+)- and the (-1 -
0013-936~95/0929-0664$09.0010 @ 1995 American Chemical Society
enantiomer, resulting in an apparent enrichment of (-)a-HCH in the digested samples. We show that degradation of HCHs in sewage sludge is mainly biological and to a lesser degree chemical. Metabolites of HCHs were not detected likely because of the small amounts accumulated due to rapid further degradation.
Experimental Section Material and Reference Compounds. Technical HCH was produced by Maag (Dielsdorf, Switzerland) in the 1950s and was now used in this study. Pure a-,,8-, and 8-HCH were from Riedel-de-Haen (Seelze,Germany), and y-HCH (lindane) was from Maag. Stock solutions were made at concentrations of 4-5 pgluL in ethyl acetate, which were then diluted according to requirements. 13C6-Hexachlorobenzene (13C6-HCB,courtesy C. Rappe, UmeA, Sweden) for use as an internal standard was made up in ethanol at a concentration of 10 nglpL. Digested Sewage Sludge. The sludge was from the anaerobic stabilizer of the communal sewage treatment plant Zurich-Glatt, Switzerland. This plant consists of mechanical and biological treatment of waste water from 100 000 inhabitants and uses mesophilic anaerobic sludge stabilization. The plant is considered to be typical of many other installations in this country and was previously studied in detail (see ref 14). The sludge contained ~ 3 of % dry matter and seemed deficient of nutrients (lack of CHI and COz evolution); it had a pH of 7.8. It was kept at 8 "C until used for the incubation experiments. Addition of starch and yeast to the sludge restarted gas production (presumably CH4 and Cod to some degree (see below). Blank determinations revealed no HCHs present (detection limit, 0.2 nglg of sludge). Incubation of HCH Isomers with Digested Sludge. Approximately 250 g of sewage sludge in a 300-mL clear glass serum bottle was fortified with 100 pL of an ethyl acetate solution containing 400-500 pg of a-HCH (experiment Sl), y-HCH (experiment S2), or technical HCH (experiment S3). The fortification levels were below the water solubilitylimits of these compounds. After athorough mixing, 1 g of soluble starch and 2.4 g of bakers' yeast dissolved in 10 mL of distilled water were added as further nutrients. The bottles were tightly capped and incubated on a horizontal shaker at 25 i 1 "C for up to 14 days in the dark. Samples were taken at different time intervals, the first one after vigorous shaking immediately after all the additions were made. A control experiment (S4) with sterilized sewage sludge was carried out with technical HCH added at the same concentration level as above. For this experiment, the sludge was treated with silver nitrate (5 ppm on dry matter) and subsequently autoclaved (130 "C, 60 min) prior to fortification, incubation, and sampling. At a later time, incubation (experiment S5) was carried out with y-pentachlorocyclohexene (y-PCCH,see below). By that time the sludge was apparently aged as indicated by a lowered pH value of 7.4, and the addition of starch and yeast did not restore gas production. Incubation of Technical HCH with Pond Water. Water from a small garden pond at Wadenswil was sampled in March 1994, and sediment particles were removed by decantation. Technical HCH (800 pg in 200 pL of ethyl acetate) was added to ~ 2 . L5 of water, and the water was exposed in a clear Pyrex glass bottle to natural sunlight at 20-25 "C for up to 26 days. A magnetic stirrer with a Tefloncoated bar prevented sedimentation of particles and newly grown algae. The bottle was loosely covered with alu-
minium foil. The pond water showed no detectable concentrations of HCH prior to fortification (detectionlimit, 0.02 nglL). Extraction and Cleanup of Sludge Samples. Samples ( ~ 2 0g) were collected in 50-mL Sovirel bottles. Immediately after collection, 10 pL of internal standard solution (100 ng of l3Cs-HCB)and 10 mL each of acetone and n-hexane were added. After being vigorously shaken, the samples were centrifuged. The yellow supematant was transferred into a 25-mL test tube and partitioned with 10 mL of distilled water. Extraction and partitioning were repeated with 10 mL of n-hexane, and the combined n-hexane layers were concentrated and passed through a small glass column containing 3 g of activated silica gel topped with anhydrous Na2S04. HCHs and other compounds were eluted with 10 mL of dichloromethane. The eluates were concentrated under a stream of nitrogen and diluted to 2-10 mL with n-hexane. Aliquots of 1pL were used for analysis by gas chromatography/mass spectrometry (GClMS). Extraction of Pond Water. Samples (50 mL) were extracted three times with 3-5 mL of diethyl ether. After being dried with anhydrous Na2S04,the combined extract was carefully concentrated and diluted to 2-10 mL with n-hexane, and aliquots of 1 pL were used for GUMS analysis. No internal standard was added. Synthesis of PCCHs. Approximately 20 mg of an HCH isomer was dissolved in x l mL of dry pyridine and heated in glass ampules for 14 h at 65 "C. The reaction mixtures were acidified with dilute ( ~ 5 %HC1 ) and partitioned with 2-3 mL of n-hexane or dichloromethane (for/3-HCH).The extracts were then washed with dilute HCl, 5% NaHC03 solution, and water. After being dried over anhydrous Na2S04,the solutions were concentrated;the residues were redissolved in n-hexane and directly analyzed by GUMS. y-PCCH was also prepared from y-HCH by treatment with NaOHltetrahydrofuran (THF) and subsequently separated from unreacted y-HCH by silica column chromatography (15).
Enantioselective HRGC/MS Analyses. A VG Tribrid double-focusing magnetic sector hybrid mass spectrometer (VG Analytical, Manchester, England) was used. A 25-m SE54 fused silica (0.32 mm i.d.1 column was used for achiral HRGC and a20-m PSO86-PMCDglass (0.30mm i.d.) column (PMCD = permethylated P-cyclodextrin; relative amount, 20%;film thickness, 0.3pm) for chiral HRGC. Samples were on-column injected at 50 "C, and the columns were temperature programmed as follows: 60 "C, 2-min isothermal, 20 "Clmin to 120 "C, then 3"lmin to 250 "C, followed by an isothermal hold at this temperature. All samples were analyzed by electron ionization (EI,70 em using selected ion monitoring (SIM) for optimal separation of HCH and PCCH isomers and enantiomers. Up to six ions were monitored simultaneously (0.5 slscan) for the detection ofHCHs and PCCHs (mlz180.938,182.935, 216.915, and 218.9121, for I3C6-HCB (mlz 287.833 and 289.830); a lockmass of m / z 207.033 from the siliconebleed of the column was used. Enantiomeric ratios (ERs) were defined as previously (16) and determined from peak area ratios assuming identical response factors for the respective enantiomers. The amount of an HCH isomer/ enantiomer in the sewage sludge samples was determined from peak area ratios relative to the internal standard, using the quantitation ions at mlz 219 and 290 for HCH and 13C6-HCB,respectively, and corrected for sample size. The concentrations (c) were then expressed in percent relative VOL. 29, NO. 3,1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
666
CHART 1
Structure of Four Most Common HCH Isomersa
CI
CI
CI
CI I
H
it
P-HCH
yHCH
I I
I
I
CI
H
6-HCH
A
, I
CI H
j
I
I
H
CI
!
H
(+)-a-HCH a
I
I
H (-)-a-HCH
The thermodvnamicallv most stable conformers are shown. The absolute configurations of
(+I- and (-)-a-HCH are from ref
19. TABLE 1
Isomer and Enantiomer Comaosition of Technical HCH and HCH Isomers Used in the Study product (WP/O)~ component a-HCH P-HCH y-HCH 8-HCH
a-HCH
B-HCH pHCH 6-HCH
99.5 (rac) x0.3 0.99). In Table 2, we list the rate constants k determined from regression plots ln(c/co)= -kt (using a no-intercept model), and the half-lives calculated as t = In 2/k. The rate constants show good agreement for (+)and (-)-a-HCH and for y-HCH when incubated either as the pure isomers (experiments S1 and S2) or the technical
y-HCH
FHCH
6-HCH
34.2 33.8 6.7 4.2 91 20.4f 0.1
3.9 0.66 4.9 93 178
5.5 1.1 4.0 80 126
product (experiment S3), indicating no influence of one isomer on the degradation of another. The rate constants varybetween3.9 x 10-3and3.4 x h-lforp- andy-HCH, the slowest and the fastest degraded isomer, respectively. The k values listed in Table 2 indicate a 2-3-fold difference among the two enantiomers of a-HCH with (+Ia-HCH degraded faster. The degradation is highly enantioselective and thus probably mainly biotic. As shown by the chromatograms in Figure 3a-d, incubation of technical HCH and of racemic a-HCH resulted in an apparent enrichment of (-)-a-HCH in the samples. After incubation for 172 h, the initial ERs of 1.00 had changed to 0.14 and 0.12 for technical HCH and a-HCH, respectively, indicating a 7-8-fold excess of (-)-a-HCH. The k values for these degradations in sewage sludge were in the order y-HCH > (+)-a-HCH > (-)-a-HCH > 8-HCH > P-HCH. This order correlates with the number of axial Cl's in an isomer: y-HCH with the maximum number (3) of axial Cl's has the fastest degradation rate, and P-HCH with no axial Cl's (all Cl's equatorial) has the slowest rate. This indicated the axial Cl's to playa dominant role in this degradation, which is consistent with antiperiplanar dehydrohalogenation or dehalogenation reaction mechanisms (see below). Interestingly, there is a linear dependence between log k and 0, the number of axial Cl's (see Figure 4; r = 0.89; correlation significant at p = 0.05) with the values for (+I-a-HCH above and for (-)-a-HCH below the regression line. During incubation of a mixture of isomers, the isomeric ratio Rise will change if the rate constants differ. In this way, the ratio y-HCHla-HCH in experiment S3 decreased from 0.175 to a-HCH > 8-HCH > P-HCH. The rates correlated with the number of axial Cl's in an isomer, consistent with anti-periplanar dehydrochlorination or dehalogenation mechanisms of these compounds. A similar order (y-HCH > a-HCH > P-HCH) was found in the degradation by rat liver cytochrome P450 in the presence and the absence of molecular oxygen (22). The degradation rates observed in active sludge are significant. As an example, the value found for y-HCH (k = 3.4 x lo-* h-l) is ~ 4 6 higher 0 ~ than the hydrolysis rate (k = 7.4 x h-l) calculated from ref 21. Degradation was significantlyslowed in sterilized sludge but still much faster than in pond water where degradation apparently was more or less by hydrolysis alone. A comparison of the rates in active and sterilized sludge indicated that degradation of HCHs in active sludge is to 80-95% biotic. The comparably still rapid degradation in sterilized sludge is considered to be caused by chemical reactions with additional, likely nucleophilic (sulfur) compounds present in the sludge, possibly in surface-catalyzed reactions with colloids and other materials present. For instance, the rate constants for the degradation of y-HCH in active sludge, in sterilized sludge, and in pond water were in the ratio 170:35:1. We also showthat isomerization
95 90 85 80 15 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0
,y
- PCCH
f
a-HCH
,8 - PCCH\
6 - PCCH
124-TCB
x2 h
7-HCH
I
4:bO
2:'OO
6
0
14100
16100
TI&
FIGURE 8. El SIM (mlz 181) chromatograms of a composite sample of synthetic PCCHs. Note the resolution of five PCCHs into pairs of enantiomers, using the PS086-PMCD HRGC column. X1 and X2 are unknown PCCHs formed as side products from a-HCH (see text). Note also the presence of a - and y-HCH and of 1,2,4-trichlorobenzene (1,2,4-TCB). r 3
100 h
80
4
.
0
5
L O 10
15
20
25
30
35
40
45
50
time ( h )
-
FIGURE 9. Simulated concentrations of educt ( G ) ,initial (q),and final metabolite (4) in sequential reactions (E- I P) as a function of time with Ukf= 40. Note that c, reaches a maximium of 2.2% ( t z 3 h) and of ~ 1 after % 28 h of incubation (right-hand scale). The kvalues used were 3.4 x lo-* and 1.35 h-lfor kf and kd, respectively.
of HCHs is of minor importance. For instance, the isomerization of y-HCH into a-HCH was ~ 2 4 slower 0 ~ than overall degradation in active sludge. Enantiomerization of a-HCH is considered to be even less important, as outlined before. Degradation of a-HCH in active sludge was enantioselective with the (+)-enantiomer degraded faster andleading to an apparent enrichment of (-1-a-HCH in incubated sludge. The rate constants for the two enantiomers were h-l/ 7.0 x in the ratio r = 1.98 x h-' = 2.82, whereas no enantioselectivitywas observed in the sterilized sludge ( r = 1.00). Since the ratio of 2.82 was produced by a consortium of microorganisms, there are likely particular strains in active sewage sludge that show even higher enantioselectivity. In experiments with sludge from the same installation taken 6 months later, a similar enantioselectivity was found ( r = 2.18; see ref 24). Microbial actions on a-HCH under aerobic conditions apparently are also enantioselective. Data from previous studies on surface waters (lakesand oceans) and laboratory
experiments with seawater under presumed aerobic conditions indicate similar trends with a faster degradation of (+I-a-HCH (10,25). The enantiomeric ratios (+I-a-HCHI (-)-a-HCH were 0.85-0.87 in the North Sea and the Baltic Sea (IO),0.75 in Canadian freshwater lakes (29,and as low as 0.4 in a glacial-fed lake of the Yukon River basin with a very long hydraulic residence time (26). Nevertheless, (+Ia-HCH is more abundant in tissue of most aquatic organisms, particularily in the warm-blooded ones (e.g., seal blubber, ER = 2-3). This can be caused by a higher accumulation of this enantiomer or by a faster metabolization of (-)-a-HCH in these organisms. However,there was one report indicating a large excess of (-) -a-HCH (ER = 0.1) in one particular seal from Murmansk, Russia (27). This excess was ascribed to a possible contamination of feed with y-HCH and its subsequent enantioselective conversion into (-)-a-HCH. In light of our data, however, there is another possible explanation. Assuming similar biochemical pathways in this seal as in other seals, an ER of 0.1 could also suggest that this particular animal received feedwith an23-35-foldenantiomeric excess of (-)-a-HCH. Racemic a-HCH thus depleted of the (+)-enantiomercould originate from an anaerobic process, as we have observed in our sewage sludge experiments. This would suggest that anaerobic degradationwas involved before this seal received its high dose of (-)-a-HCH. The degradation of HCHs in the active sludge is considered to be caused by anaerobic, methanogenic, mesophilic microorganisms that were not yet further characterized. The sludgesinvestigated were from a typical installation, representative of many other installations in this country (28). The actual rate constants in other sludges, however, may well vary and depend on the exact status of a sludge and the amounts and nature of nutrients present for the microorganisms. Since HCHs are likely degraded in cometabolic reactions, a deficiency in such nutrients would slow degradation, as we have observed in an aged sludge. VOL. 29, NO. 3. 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY 1 6 7 1
Despite extensive efforts, we were unable to detect metabolites of the HCHs in these sludge experiments. We ascribe this to the fact that the initially expected PCCHs and TCCHs are degraded even faster to further metabolites which then escaped our detection. The fast degradation of one such compound (y-PCCH) was experimentally confirmed. The data of our experiments seem to follow first-order kinetics. In enzymatic reactions, often a Michaelis-Menten type of kinetic is observedwith apparent zero-order kinetics resulting from a saturation of the enzymes with substrates (22). Apparently, the concentrations of HCHs and other substrates in our experiments were below those causing saturation of the enzyme systems. The enzymes responsible for the degradation of HCH in the active sludge seem to prefer a common structural element represented by y-HCH and (+)-a-HCH and less by (-)-a-HCH and the other isomers.
Acknowledgments We gratefully acknowledge detailed discussions and comments of H. P. Kohler and J. Zeyer of the Swiss Federal Institute of Water Resources and Water Pollution Control (ETHIFAWAG, Diibendorf and Zurich). We also thank H. P. Kohler for putting the active sewage sludge at our disposal and C. Rappe, University of UmeB, Sweden, for the gift of I3Cs-HCB.
literature Cited (1) Anonymous. The Pesticide Fact Book; Noyes Data Corp.: Park Ridge, NJ, 1985; pp 475-483. (2) Sloof, W.; Mathijsen, A. J. C. M. Integrated Criteria Document Hexachlorocyclohexane; RIVM: Bilthoven, Netherlands, 1987; Report 758473011. (3) Sittig, M. Pesticide Manufacturing and Toxic Materials Control Encyclopedia; Noyes Data Corp.: Park Ridge, NJ, 1980; pp 8993. (4) Kutz, F. W.; Wood, P. H.; Bottimore, D. P. Rev. Environ. Contam. Toxicol. 1991, 120, 14-28. (5) Rippen, G. In Handbuch Umweltchemikalien,2nd ed.; EcomedVerlag: Landsberg, GFR, 1991; Vol. 5, Chapter y-Hexachlorcyclohexan, pp 1-36. (6) Montgomery, J. H. Agrochemical Desk Reference, Environmental Data; Lewis Publishers: Chelsea, MI, 1993; pp 248-251.
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(7) Malayandi, M.; Shah, S. M.J. Environ. Sci. Health 1984,19,887910. (8) Benezet, H. J.: Matsumura, F. Nature 1973, 243, 480-481. (9) Smith, A. G. In Handbook of Pesticide Toxicology, Vol. 2; Hayes, W. J., Laws, E. R., Eds.; Academic Press: San Diego, CA, 1991; pp 794-795. (10) Hiihnerfuss, H.; Faller, I.; Konig, W. A,; Ludwig, P. Environ. Sci. Technol. 1992, 26, 2127-2133. (11) Muller M. D.; Schlabach, M.; Oehme, M. Environ. Sci. Technol. 1992, 26, 566-569. (12) Kirk, P. W. W.; Lester J. N. WaterSci. Technol. 1988,20,353-359. (13) Schroder, H. F. Water Sci. Technol. 1987, 19, 429-438. (14) Alder, A. C.; Siegrist, H.; Gujer, W.; Giger, W. Water Res. 1990, 24, 733- 742. (15) Ludwig,P. Ph.D. Dissertation, UniversityofHamburg, GFR, 1991. (16) Buser, H. R.; Muller, M. D. Anal. Chem. 1992, 64, 3168-3175. (17) Rochling, H. In Chemie der Pflanzenschutz- und Schadlingsbekumpfungsmittel, Vol. 1; Wegler, R., Ed.; Springer-Verlag: Heidelberg, GFR, 1970; pp 129-138. (18) Kirk-Othmer.Encyclopediaof Chemical Technology,3rd ed.; John Wiley and Sons: New York, 1979; Vol. 5, pp 808-813. (19) Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; John Wiley and Sons: New York, 1994; p 707. (20) Muller, M. D.; Buser, H. R. Anal. Chem. 1994, 66, 2155-2162. (21) Ngabe, B.; Bidleman, T. F.; Falconer, R. L. Environ. Sci. Technol. 1993,27, 1930-1933. (22) Beurskens, J. E. M.; Stams, A. J. M.; Zehnder, A. J. B.; Bachmann, A. Ecotoxicol. Environ. Sa$ 1991, 21, 128-136. (23) Roberts, D. V. Enzyme Kinetics, Cambridge Chemisny Texts; Cambridge University Press: Cambridge, UK, 1977; pp 12-14. (24) Muller, M. D.; Buser, H. R. Enuiron. Sci. Technol., submitted for publication. (25) Falconer, R. L.; Bidleman, T. F.; Gregor, D. J. Sci. Total Environ., in press. (26) Alaee, M.; Spencer, C.; Hauta, C.; Palmer, M. Contaminants in the Yukon River. 24th International Symposium on Environmental Analytical Chemistry, May 16-19,1994, Ottawa, Canada. (27) Hummert, K.; Luckas, B.; Buyten, J. Distribution of +/- a-HCH and PCCH enantiomers in marine organisms. In Orgunohalogen Compounds, Vol. 14; Fiedler, H., Frank, H., Hutzinger, O., ParzefaU, W., Riss, A,, Safe, S., Eds.; University of Bayreuth: Bayreuth, GFR, 1993; pp 147-150. (28) Kohler, H. P. Swiss Federal Institute of Water Resources and Water Pollution Control, Diibendorf, Switzerland, personal communication. 1994.
Receivedfor review lune 8,1994. Revised manuscript received November 10, 1994. Accepted November 16, 1994.@
ES940355G @
Abstract published in AdvanceACSAbstructs, December 15,1994.
