Environ. Sci. Technol. 1002, 26, 2127-2133
Gas Chromatographic Separation of the Enantiomers of Marine Pollutants. 4. Fate of Hexachlorocyclohexane Isomers in the Baltic and North Sea Helnrlch Huhnerfuss,* Jorn Faller, Wllfrled A. Konlg, and Peter Ludwlg Instkut fur Organische Chemie der Universitat Hamburg, Martin-Luther-King-Platz 6, D-2000 Hamburg 13,F.R.G.
Previous laboratory investigations of the degradation of a-hexachlorocyclohexane (a-HCH) and y-hexachlorocyclohexane (y-HCH) by marine microorganisms (in general an enantioselectiveprocess) have been complemented by additional laboratory measurements which include the microbial degradation of y-pentachlorocyclohexene(yPCCH), the isomerization of y-HCH to a-HCH, and the air/water transport of the first degradation products of the two HCH isomers, y-PCCH and P-pentachlorocyclohexene (0-PCCH). In addition, a crucial experiment has been performed with regard to the photochemical decomposition of a-HCH and 8-PCCH in seawater by "artificial sunlight", which turned out to be a nonenantioselective process. The laboratory results are compared with the enantiomeric excesses of a-HCH, @-PCCH,and y-PCCH of water samples obtained at 14 stations in the Baltic Sea and 7 stations in the North Sea. As a consequence, comprehensive insight into the fate of a-HCH and y-HCH in marine waters of the Baltic Sea and the North Sea was gained. Introduction Hexachlorocyclohexanes (HCH) are widely prevalent in all parts of the world's oceans. This holds, in particular, for the insecticide y-HCH (lindane) and its main technical byproduct a-HCH. Therefore, many systematic investigations related to the distribution of HCH isomers, their "sinks and sources", and the seasonal variability of their concentrations have been performed in coastal areas and in the shelf seas, e.g., the North Sea (1-4) and the Baltic Sea (5). However, more detailed questions related to the fate of the HCH isomers could not yet be answered unambiguously by means of concentration measurements. In order to fill this gap, Faller et al. (6, 7) suggested a new experimental approach which includes the determination of the enantiomeric excess of the chiral a-HCH and of the chiral degradation products of the respective prochiral and chiral HCH isomers by means of capillary gas chromatography using 8-cyclodextrin derivatives as chiral stationary phases. The authors claimed that this method allows one to distinguish between enzymatic decomposition (mostly enantioselective) and nonenzymatic, e.g., photochemical, decomposition (mostly nonenantioselective). As a consequence, it is expected to be possible to determine the contribution of microbial and photochemical processes to the decomposition of HCH isomers in the marine ecosystem. In order to verify this hypothesis, Ludwig et al. (8) performed systematic laboratory experiments with marine microorganisms. The authors were able to analyze the main degradation products of both a-HCH and yHCH, which turned out to be 8-pentachlorocyclohexene (8-PCCH) and y-pentachlorocyclohexene (y-PCCH), respectively. Furthermore, they determined the enantiomeric ratios of these two chiral degradation products. However, it has to be stressed that the experiment had been performed under laboratory conditions; in particular, photochemical processes had been carefully excluded. A systematic comparison between these laboratory experiments and the complex in-situ situation of the open sea was lacking. 0013-936X/92/0926-2127$03.00/0
In this paper, water samples of the North Sea (German Bight; Skagerrak) and of the Baltic Sea have been analyzed to determine the enantiomeric excess of a-HCH and its main degradation product 0-PCCH, as well as the main degradation product of y-HCH, i.e., y-PCCH. The results obtained will be compared with laboratory degradation measurements using a culture of marine microorganisms which is known to be representative for the German Bight and presumably also for the Baltic Sea (although the salinity of the latter is lower than that of the North Sea). The laboratory experiments reported herein have been extended in comparison to earlier results (8),now including additional pathways, Le., the air/water transport of the first degradation products y-PCCH and 0-PCCH, the degradation of y-PCCH, and the isomerization of y-HCH to a-HCH. In addition, a crucial experiment has been performed with regard to the photochemical decomposition of a-HCH and 8-PCCH in seawater, shedding light on the question of whether or not this is an enantioselective process (photochemical reaction by means of organic marine mediator compounds). As a consequence, a comprehensive survey on the fate of a-HCH and y-HCH in marine waters of the Baltic Sea and the North Sea can be given. Methods and Materials (a) Media and Cultural Conditions. A 0.5-g sample of aerobic sediment (containing organic carbon sources, mainly dead algae) and 200 mL of seawater taken off the North Sea island Heligoland (54'11' N, 7O53' E) were shaken in 500-mL laboratory flasks, closed with caps that allow the exchange of air, in the dark for 4 weeks at room temperature (291-293 K) with 0.2 mg of y-HCH (Promochem) and racemic a-HCH (Promochem), respectively, dissolved in 0.5 mL of acetone, thus giving an enrichment culture which was used in all experiments reported herein. Both the sterile controls and the degradation experiments were carried out in the following medium: 150 mL of aged seawater [station Zisch 8, 25 m; see Huhnerfuss et al. (911, 50 mL of distilled water, 1 g of yeast extract (Merck), 0.1 g of @-nicotinamideadenine dinucleotide (Fluka), and 2 mg of y-HCH and 0.4 mg of a-HCH, respectively, dissolved in 0.5 mL of acetone. For the degradation experiment, the sterile medium was inoculated with 0.2 mL of the enrichment culture described above and shaken under the same conditions as the sterile control. (b) Baltic and North Sea Water Samples. Water samples were taken at 4 stations in the German Bight area of the North Sea, 3 stations in the Skagerrak, and 14 stations in the Baltic Sea (see Figure 1). The sampling depth was 10 m, and the salinity ranged between 34% (German Bight), 31.6% (Skagerrak), and about 7.3-10950 (Baltic Sea). The water samples were extracted, purified, and analyzed according to known procedures [for details see Ernst et al. (IO) and Gaul and Ziebarth (I)],which include extraction of 10 dm3 of seawater by 200 cm3 of n-hexane, purification of the hexane solution by column column, and fractionation chromatography over an A1203 by HPLC. (c) Analytical Procedure. A 5-mL aliquot of the medium described above [see (a)] was extracted with 2 mL of distilled n-hexane (p.a., Merck) which contained 6-HCH
0 1992 Amerlcan Chemical Society
Environ. Scl. Technol., Vol. 26, No. 11, 1992 2127
A I
CI
I
Cl
1
Xb
Ia
.L I
B Flgure 1.
taken.
Posklons of the stations at which water samples have been
wcl CI
I11
c1
\
/ as an internal standard. The organic phase was centrifuged (30 min at 3500 rpm), and a 50-pL aliquot was used for the subsequent quantitative gas chromatographic analysis, applying two columns with different polarities [Mega gas chromatograph, Carlo Erba 5600 HRGC; 2-pL on-column injection; capillary columns, 30-m DB608 Supelco and 25-m NB-54 HNU-Nordion with carrier gas helium (150 kPa) and 63Ni-electron-capture detector (ECD) with 1 I 1 I make-up gas nitrogen]. In addition, the degradation products 7-PCCH and 0-PCCH were identified by gas chromatography/mass spectrometry (E170 eV; VG-Analytical VG 70-250s). Prior to the analysis of the enantiomeric ratios, the reFlgwe 2. (A) Enantiomers of a-HCH (Ia and Ib) and the correspondlng 0-PCCH enantlomers (IIa and IIb). (a) y-HCH (111) and the corremainder of the sample was cleaned by column chromaspondlng 7-PCCH (IVa and IVb) enantbmers. An asslgnment of the tography over a partially deactivated (5% water) Al,03 absolute struchre to the (4-) or (-)+nantkmer Is currently not possible. column (ICN) by elution with 5 mL of distilled n-hexane. After evaporation to 1mL, a 0.2-mL aliquot was used for Table I. Enantiomeric Ratios of @-PCCHand yPCCH isolation of the PCCH isomers and a-HCH with a LiFormed by Microbial Degradation of a-HCH and T-HCH, chrosorb 100 Si column (Merck) by HPLC (Merck/Hitachi Respectively, during a Period of 4 Weeks" L6200). The respective fractions were evaporated to 500 enantiomeric ratios pL and analyzed by using two different chiral fused-silica capillary columns, which had been specially prepared for (A)@-PCCH (B)r-PCCH expt expt investigations with an ECD [for details, see Faller et al. ( 6 ) ) column A, length 25 m, i.d. 0.25 mm, coated with time (days) I I1 I I1 heptakis(3-0-butyryl-2,6-di-O-pentyl)-~-cyclodextrin, 0 carrier gas helium (60 kPa); column B, length 25 m, i.d. 7 1.16 1.20 0.99 1.00 0.25 mm, coated with 50% heptakis(2,3,6-tri-O-n14 1.32 1.36 1.02 0.98 pentyl)-0-cyclodextrin and 50% OV1701, carrier gas he21 1.13 1.21 1.00 0.99 28 1.18 1.15 1-01 1.00 lium (45 kPa); both on a Vega gas chromatograph (Carlo Erba), 2-pL on-column injection, 63Ni-ECDwith nitrogen The data are average values of two injections. The control data as make-up gas. For retention time comparison, standard for 6-PCCHshowed values of 1.00 * 0.02 throughout the experimeasurements, and degradation experiments,7-PCCH and ment [from Ludwie et al. (8)l. j3-PCCH were prepared according to the methods of Munster et al. (11) and of Ludwig (12). the prochiral y H C H can form two 7-PCCH enantiomers. During a nonenzymatic dehydrochlorination of the HCH Results and Discussion isomers, an achiral environment is expected to produce a racemate. But in the case of an enzymatic elimination,one (a) Laboratory Experiments. Microbial Degradawould predict a preferential formation of the specific tion and Isomerization. Recently Ludwig et al. (8) PCCH enantiomers. proved that the enrichment culture described above effects This hypothesis had been investigated by Ludwig et al. a trans elimination of HC1 both in a-HCH and in 7-HCH, (8)using an enrichment culture of marine microorganisms. thus giving rise to the formation of P-PCCH and 7-PCCH, The results are summarized in Table I. While the average respectively, as schematically shown in Figure 2A and B. enantiomeric ratios of 8-PCCH (see Table I, A) changed Each of the a-HCH enantiomers can be dehydrochlofrom 1.18 at the beginning to 1.34 as a maximum value to rinated to one specific P-PCCH enantiomer only, whereas 2128
Envlron. Sci. Technol., Vol. 26, No. 11, 1992
Table 11. Enantiomeric Ratios of 8-PCCH, during a Period of Microbial Degradation of 3 weeks, and of the Sterile Control (pH 7.5)" enantiomeric ratios of 6-PCCH expt I expt I1 control
time (days) 0
1.00
1.00
1.00
3
0.99
0.97
1.00
6 9
0.85 0.77 0.71 0.63
0.83
1.00
0.79
0.99
0.76 0.71 0.51
1.00
12 16 21
0.50
0.99
1.00
Table IV. Enantiomeric Ratios of a-HCH Formed by Microbial Degradation of y-HCH, during a Period of 4 Weeks, and of the Sterile Control"
0 7
1.03 f 0.05 1.00 f 0.07
"The data are average values of three experiments, whereby each sample has been injected two times. t
L
Table 111. Enantiomeric Ratios of y-PCCH, during a Period of Microbial Degradation of 3 Weeks, and of the Sterile Control" time (days)
1.00 0.98 f 0.12 0.99 f 0.08 1.02 f 0.09 1.00 f 0.09
1.00 0.99 h 0.08 1.02 f 0.10
14 21 28
The data are average values of two injections [from Ludwig et al. (S)].
enantiomeric ratios of y-PCCH expt I expt I1 control
enantiomeric ratios of a-HCH av of expts 1-111 control
time (days)
s e
-
M
0
1.00 1.12 1.43 1.43 1.33 1.18 1.16
0 3 6 9
12 16 21
1.00 1.15 1.47 1.67 1.39 1.41 1.40
1.00 0.99
1.00 0.99
1.00 1.00 0.99
" The data are average values of two injections.
-
1.17 at the end of the experiment (enantioselectivity),they remained at 1.00 within the error limits during the whole experiment in the case of y-PCCH (see Table I, B) (no enantioselectivity). The appearance of a maximum indicates that during the first 2 weeks the formation of 0PCCH dominates, while thereafter the subsequent degradation of 0-PCCH becomes increasingly important. These results imply that, in the case of the HCH isomers, a prochiral substrate (7-HCH) is not sufficient for an enantioselective enzymatic degradation. A chiral substrate (a-HCH) is required in order to get enantioselective decomposition. In addition, Ludwig et al. (8) studied the further fate of 0-PCCH and obtained the results summarized in Table 11. It is noteworthy that the 8-PCCH enantiomer eluating first is produced by dehydrochlorination of (+)-a-HCH, while the second P-PCCH peak correlates with (-)-a-HCH. Therefore, the enantiomeric ratios summarized in Tables I and I1 imply that (+)-a-HCH and its corresponding 8PCCH enantiomer are degraded more easily than (-)-aHCH and its corresponding 0-PCCH enantiomer, and it can be assumed that the responsible enzymes prefer a common structural element represented by (+)-a-HCH and the corresponding P-PCCH enantiomer. Basically, the degradation of P-PCCH may be caused by nonenzymatic processes as well as metabolic and cometabolic microbial processes. As a result, various monochlorobenzene, dichlorobenzene, and trichlorobenzene derivatives and monochlorophenol, dichlorophenol, and trichlorophenol derivatives are formed. For details the reader should refer to ref 12 and to the summarizing Figure 6. The enantiomeric ratios of y-PCCH during a 3-week period of microbial degradation and of the sterile control are shown in Table 111. The average values of experiments I and I1 attain maximum ratios of 1.56 after -9 days and approach values of 1.28 after a period of 3 weeks. While the formation of y-PCCH by microbial degradation of
-
0
3
0
9
12
15
18
21
time [days]
Figure 3. Concentrations of sterile controls of @-PCCH(open circles) and of y-PCCH (dark squares),whlch were shaken in 500-mL laboratory fiasks, closed wlth caps that allow airlwater exchange of organic substances, during a period of 3 weeks.
y-HCH is nonenantioselective, the values summarized in Table I11 imply that the subsequent microbial degradation of y-PCCH is enantioselective. Degradation products of yPCCH include monochlorobenzene, dichlorobenzene, and trichlorobenzene derivatives and monochlorophenol, dichlorophenol, and trichlorophenol derivatives (see ref 12 and Figure 6). In addition, laboratory experiments were performed to investigate the potential formation of a-HCH either by direct isomerization of y-HCH or, alternatively, by addition reaction of HC1 with y-PCCH. The microbial isomerization reaction had been observed by Benezet and Matsumura (13)and by Malaiyandi and Shah (14),while the microbial addition reaction of HC1 to y-PCCH had been observed by Vonk and Quirijns (15). In both cases terrestric microorganisms had been responsible for these reactions. The enrichment culture of marine microorganisms used in this work was not able to convert yPCCH to a-HCH. However, the direct isomerization of y-HCH to a-HCH has been found for this enrichment culture of marine microorganisms. The enantiomeric ratios of aHCH formed by microbial degradation of y-HCH during a 4-week period are summarized in Table IV. It turned out that the enantiomeric ratios remained 1.00 within the error limits throughout the experimental period. Therefore, the conclusion drawn for the formation of yPCCH by microbial degradation of y-HCH by marine microorganisms is confirmed: In the case of the HCH isomers, a prochiral substrate (7-HCH) is not sufficient for an enantioselective enzymatic degradation. A chiral substrate, e.g., a-HCH, y-PCCH, or 8-PCCH, is required in order to get enantioselective decomposition. Water/Air Transport. In order to study the potential water/air transport of the first degradation products of a-HCH and y-HCH, two sterile controls of P-PCCH and y-PCCH, respectively, were shaken in 500-mL laboratory flasks, closed with caps that prevent the test solution from spraying but allow the exchange of air and organic sub-
-
Envlron. Scl. Technol., Vol. 26, No. 11, 1992 2120
Table V. Concentrations (ng/dma) of a-HCH, and of yHCH, the Ratios yHCH/a-HCH, and the Enantiomeric Ratios of a-HCH, ’PPCCH, and 8-PCCH As Determined in Water Samples Obtained at the North Sea and Baltic Sea Stations Shown in Figure lo
station no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
15 16 17 1Bd 19d 20d 21d
Stations 1-14 et al. (7). (I
concn (ng/dm3) r-HCH (U-HCH 5.0 4.5 4.4 4.4 3.6 3.5 7.7 4.2 4.9 5.3
4.5 5.3 4.9 5.8 5.4 4.4 5.2 5.5 4.2 4.9
r-HCH/ (U-HCH
enantiomeric ratios rl-PCCH/ 72-PCCH
&-PCCH/ PZ-PCCH
1.1 0.85 0.90 0.77 0.67 0.80 1.48 0.76 1.17 1.08
0.89 1.16 0.79 1.16 0.87 1.17 1.12 0.85 0.83 0.85 C 0.83 C 0.84 1.13c 0.85 C 0.83 0.85 2.0 0.2 10.0 0.92 2.0 0.2 10.0 0.84 0.82 1.15 0.85 av value, Baltic Sea i0.03 i0.02 0.81 0.84 0.87 2.7 1.3 2.10 0.83 2.1 2.4 0.85 0.88 1.0 1.2 0.85 0.94 2.3 2.9 0.82 0.94 0.87 av value, North Sea k0.05 Baltic Sea; stations 15-21 North Sea. Value of pooled stations 1-10. Value of pooled stations 7-1G.
stances, during a period of 3 weeks. The results, which are shown in Figure 3, show that in the case of 8-PCCH a relatively small amount has been transported into the air, while in the case of y-PCCH, more than 99% of the compound had escaped from the water. Although the experimental conditions, Le., shaking of the flask throughout the experimental period, are supposed to be much more drastic than in the open sea, the clearly different characteristics of the two curves shown in Figure 3 suggest that a significant part of the y-PCCH that is produced by microbial degradation of y-HCH is directly transferred into the air, whereas for the first microbial degradation product of a-HCH, Le., @-PCCH,air/sea exchange appears to be less important. (b) Water Samples from the Baltic and the North Seas. A typical gas chromatogram showing the enantiomeric separation of a-HCH extracted from a Baltic Sea water sample (station 5) using a fused-silica capillary column coated with 50% heptakis(2,3,6-tri-O-n-pentyl)8-cyclodextrin and 50% OV1701 is given in Figure 4. Typical gas chromatograms of the two 0-PCCH and yPCCH enantiomers extracted from Baltic Sea water samples are shown in Figure 5A (stations 1-10 pooled) and B (station 4), respectively. The concentrations of a-HCH, and of y-HCH, the ratios y-HCH/a-HCH, and the enantiomeric excesses of a-HCH, y-PCCH, and 8-PCCH of water samples obtained at 14 stations in the Baltic Sea and 7 stations in the North Sea are summarized in Table V. Concerning the Baltic Sea samples, the concentrations of yHCH and of a-HCH agree sufficiently well with earlier results of Gaul (5); i.e., the concentration values are assumed to be representative of the southern Baltic Sea. The ratios yHCH/a-HCH indicate that the water bodies were not completely uniform; however, according to Hiihnerfuss and Weber (4), the water samples exhibiting y-HCH/aHCH ratios of 100
110
120
130
time [min]
Figure 4. Enantiomeric separation of a-HCH extracted from a Baltlc Sea water sample (station 5) using a fused-silica capillary column coated with 50% heptakis(2,3,6-tri-O-n-pentyl)-~-cyclodextrinand 50% OV1701. Column temperature program: 323-10 K/min to 388 K; carrier gas, 45 kPa helium: on-column injection: ECD.
for a considerable time after input of the technical HCH mixture to the sea. The enantiomeric ratios of (+)-a-HCH/ (-)-a-HCH are relatively constant at the 14 stations of the Baltic Sea, showing an average value of 0.85 f 0.03. This value compares well with the average value of the seven North Sea stations, 0.87 f 0.05, which in turn implies that the microbial degradation pathways can be assumed to be comparable in the two sea areas investigated herein. Both the value for the Baltic Sea and for the North Sea agree with the average value of North Sea water samples obtained in the eastern part of the North Sea, the German Bight, and in the Skagerrak during the Zkch winter campaign in 1987 (0.85 f 0.1; see ref 7). Regarding the enantiomeric ratio of y-PCCH as determined from the Baltic Sea water samples, an average value
rpCcH
Bi-PCCH
\1
A
Table VI. Enantiomeric Ratios of CY-HCHand 8-PCCH Formed by Photochemical Reactions during a Period of 35 Daysa
enantiomeric ratios (+)-a-HCH/ Bi-PCCHI . . (-)-a-HCH '&-PCCH
time (days)
0.54 0.47 0.46 0.48 0.49 0.46 0.47 0.45 0.49 0.48 0.48 0.44
0 5 7 9 12 14 16 21 23 26 29 35
__t_i__k 80 90 100 time Cminl yI-PCCH
B
1.21 1.02 0.96 0.84 0.69 0.67 0.62 0.47 0.51 0.48 0.45 0.44
"At the beginning of the experiment the enantiomeric ratios for a-HCH and B-PCCH were 0.54and 1.21,remectivelv. ~~~
I
50
I
60
,
I
70
I I
80
*
time [min]
Figure 5. Chromatograms of the two &PCCH (A) and y-PCCH enantlomers (B) extracted from a BaRlc Sea water sample (A, stations 1-10 pooled; B, statlon 4) using a fused-slilca capillary column coated wlth 50% heptakls(2,3,8-trI-O-n-pentyl).~-cyclodextrln and 50% OV1701. Column temperature program: 323-10 K/min to 388 K; carrier gas, 45 kPa hellum; on-column Injection; ECD.
of 1.15 f 0.02 has been measured. In Table V, yl stands for the first peak of the two enantiomers in the gas chromatogram, while yz denominates the second peak. This value has to be compared with the corresponding average value obtained by the laboratory measurements summarized in Table 111, 1.28 f 0.12 after 3 weeks; i.e., the laboratory value is approaching the value measured in the open sea. As a consequence, the following degradation processes can be assumed: y-HCH is dehydrohalogenated by marine microorganisms nonenantioselectively, thus forming yPCCH with an enantiomeric ratio of 1/1. As recently shown by Faller (16), the same holds for photochemical degradation of y-HCH; i.e., y-PCCH is formed in the marine ecosystem both by microbial and by photochemical processes without enantiomeric excess. However, since the further microbial degradation of y-PCCH is enantioselective, the average value of 1.15 represents a mixture of nonenantioselective formation (1.00)and enantioselective degradation (1.28)of y-PCCH. At first glance, the enantiomeric ratio of 1.15 for y-PCCH seems to suggest that nonenantioselective formation and enantioselective degradation of y-PCCH in the Baltic Sea are of comparable importance. However, this is not the case, because the measurements summarized in Figure 3 clearly show that most of the y-PCCH that is formed by degradation of y-HCH is being transported from the water column into the air by air/sea exchange processes. As a consequence, a remainder of few percent of yPCCH only is available for degradation, further giving rise to the enantiomeric excess as determined in the Baltic Sea samples. The determination of the enantiomeric excess of 8PCCH in the Baltic Sea was difficult because of its low concentration. But as outlined above, the enantiomeric ratios of a-HCH were comparable in all water samples, which suggested that the microbial characteristics in these water samples appeared to be sufficiently well in accord. Therefore, it seemed acceptable to pool the water samples of stations 1-12, in order to get a reliable value for the
enantiomeric excess of 0-PCCH in the Baltic Sea. In Table V, stands for the first peak of the two enantiomers in the gas qhromatogram, while 8, denominates the second peak. At fist glance, the value of 0.97, which is close to 1.00 within the error limits, is astonishing and appears to be in contradiction both to the enantioselective microbial formation of 8-PCCH from a-HCH (see Table I) and to the subsequent enantioselective microbial degradation of j3-PCCH (see Table 11). (c) Photochemical Degradation. The missing link in the interpretation of the above enantiomeric excess of 8-PCCH has been obtained by systematic laboratory measurements which include photochemical degradation of both a-HCH and 0-PCCH. As a starting point, a mixture of the two compounds was used which had been obtained in the course of the preparation of (-)-a-HCH by reaction of technical (f)-a-HCH with (+)-brucin (17). After a first preparative cycle, the ratio (+)-a-HCH/ (-)-a-HCH is -0.54, and the ratio &-PCCH/B,-PCCH is 1.21. An enantiomeric excess of more than 90% requires at least three to four preparative cycles according to Ludwig (8). Herein, we used the mixture obtained after the first preparative cycle. As shown in Table VI, the enantiomeric ratio (+)-a-HCH/ (-)-a-HCH remains constant throughout the experimental period, exhibiting an average value of -0.48 f 0.04. The same value is approached by the ratio @,-PCCH/B,-PCCHafter a photochemical reaction of -3 weeks. This implies that the photochemical decomposition of the j3-PCCH that had been present in the starting mixture was complete after -3 weeks; however, additional 8-PCCH has been continuously formed by photochemical degradation of a-HCH nonenantioselectively. Since the a-HCH present in the mixture exhibits an enantiomeric ratio of -0.48, the nonenantioselective formation of 8-PCCH must in this case also give rise to an enantiomeric ratio of -0.48. In summary, the photochemical degradation of a-HCH and 8-PCCH is effected nonenantioselectively; however, enantiomeric excesses of 8-PCCH that may have been formed by enzymatic processes may be modified by photochemical processes provided that enzymatic processes become less important, e.g., due to seasonal variations of microbial activity. The laboratory investigations of microbial and photochemical degradation allow the following tentative explanations for the enantiomeric excesses of a-HCH and j3PCCH determined in the Baltic Sea: An enantiomeric ratio of (+)-a-HCH/(-)-a-HCH = 0.85 indicates that
o1
Environ. Scl. Technoi., Vol. 26, No. 11, 1992 2131
evapor.
A trnosphere
evapor.
Water
MCB 12DCB 13DCB 14DCB 123TCB 124TCB 135TCB 2CP
MCB 1ZDCB 13DCB 124TCB 3CP 4CP 24DCP DCP
24DCP
11
2CP 3CP
4cp
345TCP
degradation
degradatlon
Flgure 6. Schematic presentatlon of the fate of y H C H and a-HCH as concluded from the laboratory microblal degradatlon experiments.
microbial decomposition of a-HCH must play an important role, because an exclusive photochemical decomposition of a-HCH would supply no enantiomeric excess (enantiomericratio, 1.00),as encountered at the inflow and input sites of technical a-HCH. The importance of microbial degradation processes can also be noted when the enantiomeric excess of the first degradation product, 0PCCH, is considered. Based upon the equilibrium a-HCH value of 0.85, which appears to be relatively constant over years, exclusive further photochemical degradation would lead to an enantiomeric ratio of 0.85 both for a-HCH and for 0-PCCH. Moreover, microbial decomposition of 0PCCH would shift the enantiomeric excess to even lower values, because &-PCCH is degraded faster than &PCCH. Therefore, the enantiomeric excess of 0.97 for 0-PCCH must reflect the microbial degradation of a-HCH, which preferentially supplies &-PCCH giving rise to enantiomeric ratios for 0-PCCH of up to 1.17. On the other hand, it can be argued as well that this latter value of 1.17 has never been observed in the open sea, and therefore, a considerable contribution of photochemical decomposition of aHCH and/or the preferential microbial decomposition of &-PCCH may be the cause for the observed enantiomeric excess for 0-PCCH; Le., considerable contributions of microbial degradation of a-HCH and 0-PCCH and photochemical degradation of a-HCH may explain the value of 0.97 for P-PCCH. The laboratory results strongly support the hypothesis that all three degradation processes discussed above are of comparable importance for the degradation of a-HCH in the open sea.
Conclusions The prochiral y-HCH is decomposed by marine microorganisms to y-PCCH or isomerized to a-HCH nonenantioselectively. However, the further degradation of the chiral y-PCCH is effected enantioselectively. a-HCH and b-PCCH are decomposed by marine microorganisms enantioselectively. These results imply that, in the case of 2132 Environ. Sci. Technoi., Voi. 26, No. 1 1 , 1992
HCH isomers, a prochiral substrate (7-HCH) is not sufficient for an enantioselective microbial degradation or isomerization; a chiral substrate (a-HCH, 0-PCCH, yPCCH) is required in order to get enantioselective decomposition. Measurements of the air/water transport of y-PCCH and 0-PCCH, respectively, showed that more than 99% of the y-PCCH that had been formed by degradation of y-HCH escaped from the water into the air, while in the case of 0-PCCH, this process can be neglected. Photochemical degradation of a-HCH and of 0-PCCH in seawater by “artificial sunlight” is nonenantioselective. However, enantiomeric excesses of 0-PCCH that may have been formed by enzymatic processes may be modified by photochemical processes provided that enzymatic processes become less important, e.g., due to seasonal variations of microbial activity. Determination of the enantiomeric excess of a-HCH, y-PCCH, and P-PCCH in 7 water samples of the North Sea and 14 water samples of the Baltic Sea supplied the following average values: North Sea, (+)-a-HCH/(-)-aHCH = 0.87; Baltic Sea, (+)-a-HCH/(-)-a-HCH = 0.85, yl-PCCH/yz-PCCH = 1.15, and pl-PCCH/p2-PCCH = 0.97. These values can be explained by assuming significant contributions from both enzymatic (enantioselective) and nonenzymatic (nonenantioselective) processes. A summarizing schematic presentation of the various processes contributing to the degradation of a-HCH and y-HCH is given in Figure 6.
Acknowledgments Some of the water samples were obtained with the help of members of the St. Ansgar School, Hamburg, FRG, during a cruise at the Baltic Sea. This is gratefully acknowledged. Registry No. I, 319-84-6; 11, 54083-25-9; 111, 58-89-9; IV, 319-94-8.
Literature Cited Gaul, H.; Ziebarth, U. Dtsch. Hydrogr. 2. 1983, 36, 191. Ernst, W.; Boon, J. P.; Weber, K. In Pollution of the North Sea: An Assessment; Salomons, W., Bayne, B., Duursma, E., Foerstner, U., Eds.; Springer Verlag: Berlin, 1988; p 284. Weber, K.; Balint, U.; Huhnerfuss, H. Alfred-WegenerInstitut, Bremerhaven, (FRG),Zisch experiment, unpublished results, 1986. Hiihnerfuss, H.; Weber, K. Institute of Organic Chemistry, University of Hamburg, Hamburg, FRG, Zisch experiment, unpublished results, 1986. Gaul, H. Uberwachung des Meeres, Report of the DHI; BSH: Hamburg, FRG, 1988; Part 11. Faller, J.; Hlihnerfuss, H.; Kanig, W. A,; Krebber, R.; Ludwig, P. Environ. Sci. Technol. 1991,25, 676. Faller, J.; Huhnerfuss, H.; Kanig, W. A.; Ludwig, P. Mar. Pollut. Bull. 1991, 22, 82. Ludwig, P.; Htihnerfuss, H.; Kanig, - W. A.; Gunkel, W. Mar. Chem.-1992,38, 13. Hiihnerfuea, H.;Dannhauer,H.; Faller, J.; Ludwig, P. Dtsch. Hydrogr. 2. 1990,43, 253. Ernst, W.; Schaefer, R. G.; Goerke, H.; Eder, G. 2.Anal. Chem. 1974,272, 358. Munster, J.; Hermann, R. S.; Koransky, W.; Hoyer, G. Hoppe-Seyler’s 2. Physiol. Chem. 1975, 356, 437. Ludwig, P. Ph.D. Dissertation, University of Hamburg at Hamburg, FRG, 1991. Benezet, H. J.; Matsumura, F. Nature 1973,243, 480. Malaiyandi, M.; Shah, S. M. J. Environ. Sei. Health 1984, A19, 887. Vonk, J. W.;Quirijns, J. K. Pestic. Biochem. Physiol. 1979, 12, 68.
Environ. Sci. Technol. 1992, 26,2133-2141
(16) Faller, J. Ph.D. Dissertation, University of Hamburg at Hamburg, FRG,1992. (17) Cristol, J. S.J. Am. Chem. SOC.1949,71, 1894.
Received for review December 26, 1991. Revised manuscript
received May 26,1992. Accepted June 17,1992. This work has been supported by the Ministry of Science and Technology of the Federal Republic of Germany (BMFT Project MFU 0620 “Prozesse im Schadstoffkreislauf Meer- Atmosphdre: Okosystem Deutsche Bucht”).
Reduction of Substituted Nitrobenzenes in Aqueous Solutions Containing Natural Organic Matter Frank M. Dunnivant and RenQ P. Schwarrenbach* Swiss Federal Institute of Technology, Zurich (ETHZ), Switzerland, and Federal Institute for Water Resources and Water Pollution Control (EAWAG), 8600 Dubendorf, Switzerland
Donald L. Macalady Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, Colorado 80401
Natural organic matter (NOM) from a variety of sources has been shown to mediate the reduction of substituted nitrobenzenes in aqueous solution containing hydrogen sulfide. Pseudo-first-order rate constants were proportional to NOM concentrations and increased with increasing pH and decreasing reduction potential (&) of the solution. At fixed pH and &, the carbon-normalized rate constants (kNOM)of a given compound varied less than 1 order of magnitude among NOMS derived from various natural waters. The effect of substituents on the reaction rate could be described by a linear free energy relationship (LFER) of the general form log k N o M = aEh1’(ArN02)+ b, where Eh1’(ArNO2)is the one-electron reduction potential of the nitroaromatic compound. Such LFERs were applied successfully to predict the kNoM values of previously untested compounds. The results of this study suggest that hydroquinone moieties within the NOM may play a pivotal role in the mediation of electron-transfer reactions involving organic pollutants. Introduction Abiotic reduction of organic pollutants in reducing environments (e.g., sediments, aquifers, and hazardous waste sites) has recently drawn considerable interest, since it has been recognized that such reactions can lead to transformation products of similar or even greater concern than the parent compounds ( I ) . Reactions of most interest include reductive dehalogenation of polyhalogenated hydrocarbons ( 2 , 3 ) ,reduction of nitroaromatic compounds (2,4-f?),and reduction of azo compounds (9). Some advances in understanding the mechanisms and pathways of such reactions in homogeneous aqueous systems have been achieved using model reductants (e.g., hydroquinones, iron porphyrins, and other transition metal complexes). However, previous investigations utilizing environmental matrices (e.g., sediment and dissolved and sorbed organic carbon) have not adequately provided a process-level understanding of key factors controlling the reduction reactions. One important and largely unanswered question is, which natural reductants are involved in the abiotic reduction of organic pollutants. Aside from biological electron donors, the most abundant natural reductants present in anaerobic environments include reduced inorganic forms of iron and sulfur [such as iron(II/III) oxides, iron(I1) carbonates, iron(I1) sulfides, and hydrogen sulfide]. Although it has been shown in various studies that these reductants react with organic pollutants (IO-I4),the reported reaction rates are often too slow to account for the 0013-936X/92/0926-2133$03.00/0
much faster transformation rates observed in some natural systems. For example, for the reduction of the nitro group of parathion or methyl parathion in anaerobic soils and sediments, half-lives of as short as a few minutes have been observed (5,6,15). Hence, there must be other, much more reactive reductants available. These highly reactive species may not be present in large abundance but may play the role of electron-transfer mediators as illustrated by Figure 1. Thus, after electron transfer to the pollutant, the mediators may be rapidly reduced again by the bulk reductants present. Possible mediators include quinone-type compounds and a variety of transition metal complexes. Such species are not only well-known components of biological electron-transfer systems (16)but they are also very likely to exist as constituents of natural organic matter (17-20). The reduction potential of natural aqueous systems is generally controlled by microorganisms, thus “bulk” electron donors and reduced forms of electron-transfer mediators may also be replenished through microbial processes. In this paper, we report the effect of natural organic matter (NOM) on the reduction kinetics of a series of monosubstituted nitrobenzenes in aqueous solutions containing hydrogen sulfide as the bulk electron donor. The major goals of this investigation were (1)to evaluate and quantify the effect of organic matter from different natural sources on the reduction kinetics of nitroaromatic compounds in aqueous solution, (2) to derive relationships for quantification of the effect of substituents on the rate of reduction of the nitro group, and (3) to gain insights into the type of structural moieties within the NOM that may be involved in mediating the reduction reactions. The results of this investigation are interpreted in light of the results of our previous investigation (8),which used two hydroquinones and an iron porphyrin as model electrontransfer mediators. Experimental Section Chemicals. Nitrobenzene (NB), 2-chloro-, 3-chloro-, and 4-chloronitrobenzene (ClNB); 2-methyl-, 3-methyl-, and 4-methylnitrobenzene (MeNB); 2-chloro-, 3-chloro-, and 4-chloroaniline; 2-methyl-, 3-methyl-, and 4-methyl(juglone); and 2aniline; 8-hydroxy-1,4-naphthoquinone hydroxy-1,4-naphthoquinone(lawsone) were purchased from Fluka AG (Buchs, Switzerland). 2,4-Dinitrotoluene, 2-nitrophenol(2-W);6-methyl-2,4dinitrophenol(DNOC); 2-nitre, 3-nitre, and 4-nitroacetophenone(AcNB); Methyl Red [2-carboxy-4’-(dimethylamino)azobenzene]; 2-amino-, 3-amino-, and 4-aminoacetophenone; nitrosobenzene; and
0 1992 American Chemical Society
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