Environ. Sci. Technol. 1991, 25, 676-678
Acknowledgment in Memorium. This paper is dedicated to Ernie Hughes, who pioneered the scientific development of the NIST gas SRM program, and who contributed so much of his technical knowledge to numerous interactions with experts in the gas analysis field, worldwide. In the present study, Ernie carried out the analyses of the NPL standards and supplied the SRMs that were analyzed by NPL, but passed away before this paper could be written. His unique insight into the requirements for accuracy in gas standards and trace gas analysis will be sorely missed by his many friends and scientific colleagues both in the United States and abroad. Registry No. CO, 630-08-0; COz, 124-38-9.
Literature Cited (1) Davenport, A. J.; Freshwater, F.; King, J. H.; Merrifield, D. R.; Partridge, R.H.; Woods, P. T. NPL Quantum M e trology Report: Analyses o f Reference Gas Mixtures f o r the Community Bureau of Reference, Commission o f European Communities; January 1985. (2) Zielinski, W. L., Jr.; Hughes, E. E.; Barnes, I. L.; Elkins, J. W.; Rook, H. L. High Accuracy Standards and Reference Methodology for Carbon Dioxide i n Air; Joint DOE/NBS Report, DOE/PR-06010-31, Technical Report TR033, June 1986.
Received for review January 4,1990. Revised manuscript received March 26, 2990. Accepted October 4 , 1990.
Do Marine Bacteria Degrade a-Hexachlorocyclohexane Stereoselectively? Jorn Faller, Heinrich Huhnerfuss, * Wilfried A. Konig, * Ralph Krebber, and Peter Ludwig Universitat Hamburg, Institut fur Organische Chemie, Martin-Luther-King Platz 6, D-2000 Hamburg 13, Germany
The enantiomeric ratio of a chiral organic pollutant has been determined gas chromatographically for the first time at low concentrations as encountered in a North Sea water sample by using heptakis(3-0-butyryl-2,6-di-O-pentyl)-Pcyclodextrin as a chiral stationary phase. As an example, the separation of the enantiomers of a-hexachlorocyclohexane (0-HCH) is shown herein. However, the method is expected to be generally applicable to many environmental problems that are related to chiral biogenic and anthropogenic substances and chiral degradation products. The potential of this experimental approach for a discrimination between enzymatic and nonenzymatic processes in marine and terrestric ecosystems is discussed. Introduction Concentration measurements of hexachlorocyclohexane isomers (HCH) in the North Sea have been performed for more than one decade, thus allowing insight into the distribution and the seasonal variability of these toxic compounds in marine waters ( I , 2). However, more detailed questions related to the fate of the HCH isomers could not yet be answered unambiguously by means of concentration measurements. In particular, information about the degradation pathways and the factors that dominate the degradation and/or isomerization of HCH isomers prevalent in the marine ecosystem is rare. Herein, a new experimental approach is suggested, which is expected to offer a possibility of distinguishing between microbial decomposition (mostly enantioselective) and nonenzymatic, e.g., photochemical decomposition (nonenantioselective) by measuring the enantiomeric excess of a-HCH in seawater, the enantiomers of which can be separated gas chromatographically by using heptakis(3-0-butyryl2,6-di-O-pentyl)-P-cyclodextrin as a chiral stationary phase ( 3 , 4). The experimental approach suggested herein is expected to be applicable to many environmental analytical problems provided that organic pollutants and their decomposition products are chiral. In the case of the eight HCH isomers, only a-HCH is chiral, exhibiting two enantiomers (see Figure l ) ,while the insecticide y H C H (lindane) is optically inactive Technically produced a-HCH should show an enantiol..dric ratio of 1:l. The same enantiomeric ratio of 1:l would have to be expected in the marine environment, if the decomposition and/or isomerization of 676
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a-HCH and y H C H were dominated by nonenzymatic processes, because the HCH isomers are exclusively manmade, and thus the enantiomeric ratio at the “sources” basically should be identical with that of technical a-HCH, and nonenzymatic processes are not assumed to lead to an enantiomeric excess of chiral compounds (nonenantioselective process). If microbiological processes are, however, dominating the decomposition of HCH isomers-be it in seawater or in terrestrial ecosystems-an enantiomeric excess can be expected, because the decomposition by microorganisms in general proceeds enantioselectively. Methods and Materials In order to distinguish between the two decomposition possibilities discussed above, water samples were collected from that part of the North Sea known to exhibit maximum a-HCH concentrations ( I ) . This investigation was part of the ZISCH experiment, which covered the complete North Sea area and included a cruise during both summer and winter seasons. First results of the ZISCH experiment allow insight into the distribution of the HCH isomers in the North Sea. In particular, the plume of the river Elbe and the outflow out of the Baltic Sea in the Skagerrak proved to be the areas with the maximum concentration of HCH isomers, while on the other hand, the area in the Central North Sea and in the Fair Isles area in the northwestern North Sea exhibited low HCH concentrations. On the basis of these ZISCH results and on the assumption that maximum concentrations are favorable to an enantioselective decomposition of a-HCH or isomerization of y H C H to a-HCH by microorganisms, the water sample investigated herein was taken in the Skagerrak south of the Norwegian coast at station 119 of the ZISCH experiment performed during the period January to March 1987. At this station the concentrations of aHCH and y H C H were 2.4 and 2.0 ng/dm3, respectively. The water samples were extracted, purified, and analyzed according to known procedures [for details see Ernst et al. (5) and Gaul and Ziebarth ( 6 ) ] ,which include extraction of 10 dm3 of seawater by 200 cm3 of n-hexane, purification of the hexane solution by column chromatography over an A1,03 column, concentration to 20 mm3 (20 ILL), and subsequent on-column gas chromatographic analysis using a 63Ni electron capture detector [ECD; 2 mm3 (2 pL) injected; isothermal 423 K; carrier gas, 100 kPa
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I
H I H
“
c1 H
H
C’
c1
J c1
c1
.
c1
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Flgure 1. Enantiomers of a-HCH, a chiral substance. No enantiomers exist for y-HCH.
helium]. It should be noted that the isothermal GC temperature of 423 K causes the retention scale to extend over 3 h. However, it turned out that this is the optimum temperature when working with such low concentrations that application of an ECD is required. A t considerable larger temperatures as, e.g., 443 K “bleeding of the column” would give rise to a considerably noisier base line. These problems are not encountered when working with “FID concentrations”. The glass capillary column, which has been specially prepared for environmental samples containing only low concentrations of pollutants (in the case of marine water samples typically of the order of nanograms per cubic decimeter of seawater), was coated with heptakis(3-0butyryl-2,6-di-0-pentyl)-/3-cyclodextrin. The procedure usually applied for coating Pyrex glass capillaries ( 3 , 4 )had to be modified for this investigation, in order to fulfill the requirements of trace analysis. In particular, emphasis had to be placed upon the purity of solvents, reagents, and glass. Furthermore, n-pentane instead of dichloromethane had to be used as a solvent for the chiral stationary phase during the coating procedure. The assignment of the (+)-a-HCH and the (-)-a-HCH enantiomers was achieved by injecting a sample of (-)-aHCH that had been prepared by the reaction of the racemate of a-HCH with brucine according to the method described by Cristol (7). Results and Discussion In order to get a reference value for the enantiomeric ratio in technically produced a-HCH, 2 mm3 (2 pL) of a standard solution of 30 pg of technical a-HCH/dm3 hexane was injected 15 times. A typical chromatogram of this series is given in Figure 2. The retention times of the two enantiomers of a-HCH are 124.8 and 133.1 min, respectively, while the peak of y-HCH appears after a retention time of 191.3 min. The average ratio of the a-HCH enantiomers as obtained from the reference chromatograms was determined to be [ (+)-a-HCH]/ [ (-)-a-HCH] = 1.03 f 0.06, which agrees sufficiently well with the theoretical value of 1 within the error limits of this method. Hence, each peak represents 30 pg of the respective a-HCH enantiomer. After the above test and reference measurements were successfully performed with technical a-HCH, the method was applied to marine water samples collected a t station 119 in the Skagerrak North Sea area. A typical chromatogram of this series is given in Figure 3, which shows that it is possible to separate the a-HCH enantiomers a t low concentrations as encountered in the marine environment. In this case, the ratio [(+)-a-HCH]/[(-)-a-HCH] was 0.85, which implies that the peak area of the (+)-enantiomer represents 20.4 pg, while the peak area of the (-)-enantiomer represents 25.4 pg. Three replications of the measurement supplied the following ratios of the two a-HCH enantiomers: 0.85, 0.89, and 0.89. The average value of this series was 0.88 f 0.03. Hence, it can be safely concluded that the average ratio of the two a-HCH enantiomers in a technical mixture (1.03 f 0.06) is signifi-
I- .
I 0
I
c
50
100
150
2 0 0 [mi111
Flgure 2. Enantiomeric separation of technical a-HCH using a 60-m glass capillary with heptakis(3-O-butyryl-2,6di-O-pentyl~(?-cyclodextrin. Column temperature, 423 K isothermal; carrier gas, 100 kPa helium; on-column injection; ECD detector.
y-H,CH K-HCH
I 0
so
100
150
200
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Figure 3. Enantiomeric separation of a-HCH extracted from a North Sea water sample (station 119; Skagerrak) using a 60-m glass capillary with heptakis(3-O-butyryl-2,6-di-O-pentyl)-(?-cyclodextrin. Column temperature, 423 K isothermal; carrier gas, 100 kPa helium; on-column injection; ECD detector.
cantly different from the ratio of the two a-HCH enantiomers in the marine water sample taken in the Skagerrak North Sea area (0.88 f 0.03). Based upon the assumption that enzymatic processes only can give rise to an enantiomeric excess, the title question of whether marine bacteria degrade a-HCH stereoselectively can be affirmed. However, it cannot be ruled out that in addition y-HCH can be transformed enantioselectively to one of the a-HCH enantiomers by microbiological action. The latter hypothesis is supported by data reported by Benezet and Matsumura (8) and by recent results of Weber et al. ( I ) and of Huhnerfuss and Weber (9), who supplied experimental evidence that in addition to the river input the transformation of y-HCH to a-HCH may be a source for a-HCH in the North Sea. Furthermore, results by Malaiyandi and Shah (IO),who discussed several possibilities for a transformation from y-HCH to a-HCH, are in line Environ. Sci. Technol., Vol. 25, No. 4, 1991 677
Environ. Sci. Technol. 1991,25,678-683
with this latter assumption. In order to verify the above hypotheses, model experiments are currently being performed in our laboratories, which will allow a decision on which of these two microbiological degradation pathways discussed above gives rise to the observed enantiomeric excess of tu-HCH. Furthermore, representative water samples from all parts of the North Sea have to be analyzed, in order to determine whether high concentrations of a-HCH and/or y-HCH are vital for the enantioselective microbial decomposition and which factors cause the enantiomeric excess. In conclusion, a modification of the enantioselective capillary gas chromatography method by Konig et al. (3, 4 ) renders it possible for the first time to determine the enantiomeric excess of chiral compounds a t low concentrations as encountered in environmental samples. Thus, a new experimental approach can be suggested that allows a discrimination between microbiological decomposition (enantioselective) and nonenzymatic processes (nonenantioselective). Acknowledgments The investigation reported herein could not have been accomplished without the dedicated assistance of numerous collegues. During the cruise, M. Gonzales-Davila, Dr. Lange, and Dr. Lutz assisted in taking the water samples, Dr. Brockmann, Dr. Huber, Dr. Kattner, and Dr. Schmidt took care of the overall logistics of the campaign. During the chemical analysis in the laboratory and the
evaluation of the data sets, valuable assistance by H. Dannhauer and H. Nommsen was provided. This is gratefully acknowledged. Thanks are also due to M. Richters, who prepared the chiral Pyrex glass capillary column. Registry No. (-)-cu-HCH, 119911-70-5; (+)-a-HCH, 11991169-2; water, 7732-18-5.
Literature Cited (1) Weber, K.; Balint, U.; Huhnerfuss, H. Mar. Ecol. Prog. Ser.,
submitted for publication. (2) Huhnerfuss, H.; Weber, K. Mar. Ecol. Prog. Ser., submitted for publication. (3) Konig, W. A.; Krebber, R.; Mischnick, P. J . High Res. Chromatogr. 1989, 12, 732. (4) Konig, W. A. Nachr. Chem., Tech. Lab. 1989, 37, 471. (5) Ernst, W.; Schaefer, R. G.; Goerke, H.; Eder, G. Z. Anal. Chem. 1974, 272, 358. (6) Gaul, H.; Ziebarth, U. Dtsch. Hydrogr. Z. 1983, 36, 191. ( 7 ) Cristol, S.J. J. Am. Chem. Sac. 1949, 71, 1894. (8) Benezet, H. J.; Matsumura, F. Nature 1973, 243, 480. (9) Huhnerfuss, H.; Weber, K. J. Geophys. Res., submitted for publication. (10) Malaiyandi, M.; Shah, S.M. J . Environ. Sci. Health 1984, A19, 887.
Received for review September 6, 1990. Accepted October 29, 1990. This work has been supported by the Ministry of Science and Technology of the Federal Republic of Germany ( B M F T projects M F U 0545 Zirkulation und Schadstoffumsatz in der Nordsee and MFU,, 0620 Prozesse i m Schadstoffkreislauf Meer-Atmosphare; Okosystem Deutsche B u c h t ) .
Measurement and Prediction of Copper Ion Activity in Lake Orta, Italy Marina Camusso and Gianni Tartari Istituto di Ricerca sulle Acque, C.N.R., 20047 Brugherio, Italy
Albert0 Zirino" Naval Ocean System Center, Code 522, San Diego, California 92152
A commercial Cu ion selective electrode (ISE) mounted on a field conductivity, temperature, depth probe (CTD) equipped with pH and oxygen sensors was used to measure a profile of Cu ion activity [a(Cu2+)]in Lake Orta, Italy. Lake Orta water contains approximately 32-34 pg L-' Cu from anthropogenic sources. Below the mixed layer, a(Cu2+)was directly related to the pH of the lake water. In the body of the hypolimnion, measurements of a(Cu2+) were in good agreement with estimates of a(Cu2+)obtained from total Cu concentrations. The pH dependence of the activity/concentration of free Cu2+was modeled with a simple ion association model of the lake water. The results of the model were verified by a potentiometric titration of a sample of lake water using Cu, pH, and NH3 ISEs. The titration simulated a forthcoming chemical treatment now in progress. Introduction Lake Orta, the seventh largest lake in Italy, occupies the southwestern part of the Lake Maggiore drainage basin. Like other glacially carved, subalpine lakes in northern Italy, it is long and narrow, extending 12.6 km north to south, with a maximum width of 1.9 km and an average depth of 70 m (Figure 1). Its important morphological characteristics are given in Table I. 678
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Table I. Main Morphometric Features of Lake Orta watershed area, km2 lake surface, km2 mean lake level altitude, mas1 mean lake length, km maximum width, km maximum depth, m mean depth, m volume, m3 theoretical renewal time, year mean water residence time, year
116.0
18.2 290 12.6 1.9
144 70 1.3 x 109
8.9 10.7
From 1926 to 1980, Lake Orta received industrial discharge containing large quantities of ammonium and copper sulfate from a plant engaged in the manufacture of rayon fiber. The discharge eliminated the floral and faunal populations of the lake and caused similar drastic changes in its chemical composition. These environmental alterations along with occasional reports on the chemical and biological conditions of the lake have been documented in many publications (1-6). In 1958, the copper load of the plant's effluent was effectively reduced from 40-80 t year-l to 4-5 t year-' and the ammonium load was reduced to 1'7'0 of its former value (2.0 X lo3 t year-') in 1981 (2). However, from the 1950s, numerous small electroplating factories distributed around the lake have continued to discharge heavy metals, al-
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