Identification of Chlorinated Dimethoxystilbene Isomers and

Anal. Chem. 2000, 72, 4859-4864

Identification of Chlorinated Dimethoxystilbene Isomers and Homologues in Bleached Paper Products Jeffrey G. McDonald and Ronald A. Hites*

School of Public and Environmental Affairs, and Department of Chemistry, Indiana University, Bloomington, Indiana, 47405

We report here the identification of a novel class of compounds, the chlorinated dimethoxystilbenes (pinosylvins), in bleached paper products. Pinosylvins are naturally occurring compounds in wood that deter infections and predators. These compounds are being chlorinated during the pulp bleaching process. Two dichlorinated pinosylvins have previously been identified in bleached paper pulp, but we have identified other isomers and more highly chlorinated homologues in various bleached paper products. These compounds are present at concentrations on the order of hundreds of ppb. On the basis of the mass spectra of synthesized standards, we can distinguish the isomers in which the chlorines are located on the ethylene moiety from those in which the chlorines are located on the aromatic ring with the methoxy groups. In addition, we can predict the chlorine substitution patterns on both rings for these compounds using linear relationships between retention indexes and calculated dipole moments. The toxicity of these compounds is not known; however, isomers related to the pesticide methoxychlor are proestrogenic.

phenols, terpenes, fats, and waxes are present in this stream. Some byproducts are recovered from this “black liquor”, and the remainder is then concentrated and burned to recover energy and inorganic compounds. Modern paper mills are also using mechanical forms of pulping, which reduces waste generated by chemical pulping. If a white paper product is needed, the pulp is bleached using a variety of processes, mixed with additives (such as optical brighteners), and formed into paper. While most of the byproducts are removed during the pulping process, some remain in the pulp and can be chlorinated during bleaching.1 Although pulp and paper mills have drastically reduced the generation of chlorinated compounds by better engineering controls and by no longer using elemental chlorine during bleaching, it is still possible that chlorinated organic molecules are present in the pulp.2 We speculated that some of these chlorinated compounds become incorporated into the bleached paper products themselves. A corollary is that there would be few if any chlorinated compounds in nonbleached paper products. Thus, to determine whether chlorinated byproducts of bleached pulp production are present in paper products, we analyzed bleached and nonbleached sheet paper, paper towels, and coffee filters from various companies.

The ubiquitous presence of anthropogenic, chlorinated organic chemicals in the environment has been studied intensely for several decades. Often these compounds are released into the environment inadvertently, for example, by chemical spills or by the improper disposal of chlorinated solvents. Other chlorinated compounds have been released intentionally, for example, by spraying to control pests. Still others have been released as the byproducts from various manufacturing processes. For example, pulp and paper mills can inadvertently produce and release a wide variety of chlorinated organic compounds.1 It is this manufacturing process that is the focus of this paper. To understand why chlorinated compounds are produced during the making of paper, a brief description of the process is helpful. The production of paper starts with the generation of pulp from wood, a process in which wood is treated with a solution of NaOH and Na2S or with Na2SO4 at elevated temperatures to break down the lignin that binds the wood’s cellulose fibers to one another. This process creates a waste stream, termed “black liquor”, containing degraded lignin and water-insoluble compounds. A wide variety of small organic molecules including

EXPERIMENTAL SECTION Paper Extractions. Four different brands of nonrecycled white sheet paper, one brand of nonbleached butcher paper, one brand each of white and brown coffee filters, and one brand each of white and brown paper towels were selected for qualitative analyses. Approximately 100 g of each product was Soxhlet extracted for 4 h with 300 mL of methanol and then for 20 h with 300 mL of dichloromethane. One hundred milliliters of water and 50 mL of saturated saltwater were added to the methanol fraction, which was than extracted with 100 mL of hexane three times. The dichloromethane fraction was reduced in volume using a RapidVap apparatus (Labconco, Kansas City, MI) to ∼3 mL and combined with the hexane from the methanol back extraction. This combined extract was solvent exchanged into hexane and reduced in volume to ∼1 mL. All extracts were fractionated on 30 cm × 1.5 cm i.d. columns of layered activated silica (Davidson Chemical, Baltimore, MD) over alumina (1% deactivated, Brockman activity I, Costa Mesa, CA) with 50 mL of hexane, 175 mL of 15% dichloromethane in hexane, and 50 mL of 40% dichlo-

* Corresponding author: (e-mail) [email protected]. (1) Kringstad, K. P.; Lindstrom, K. Environ. Sci. Technol. 1984, 18, 236-247A.

(2) McDonough, T., J. Forward. In Chlorine and Chlorine Compound in the Paper Industry; Turoski, V., Ed.; Ann Arbor Press: Chelsea, MI, 1998; pp ix-xii.

10.1021/ac000474g CCC: $19.00 Published on Web 09/15/2000

© 2000 American Chemical Society

Analytical Chemistry, Vol. 72, No. 20, October 15, 2000 4859

Figure 1. Structures of methoxychlor olefin (A), methoxychlor (B), three synthesized dichloropinosylvin methyl ethers (C, D, and E), and diethylstilbestrol (DES) (F). Retention indexes for compounds A and C-E are shown in parentheses.

romethane in hexane. Each fraction was solvent exchanged into hexane using a RapidVap apparatus and reduced in volume to ∼1 mL. The 40% fraction, which contained chlorinated compounds as indicated by preliminary GC/MS analysis, was further reduced in volume to ∼100 µL under a gentle stream of nitrogen. Extracts were transferred to GC autoinjector vials containing 100-µL inserts. For quantitative analyses, approximately 100 g of sheet paper, 60 g of coffee filters, or 40 g of paper towels was each loaded into two Soxhlet extractors (for example, 200 g of total sheet paper split between two Soxhlet extractors), spiked with p,p′-methoxychlor olefin (see Figure 1, A; AccuStandard Inc., New Haven, CT) as an internal standard and extracted for 24 h with 300 mL of 50% dichloromethane in hexane. The duplicate extracts were combined, solvent exchanged into hexane using a RapidVap apparatus, and reduced in volume to ∼3 mL. The extracts were filtered through a funnel tightly plugged with glass wool and filled with anhydrous Na2SO4. The extracts were washed 3-6 times with 100 mL of water until no turbidity was observed. Fifty milliliters of saturated saltwater was added to the 300-600 mL of wash water from the previous extraction, and this solution was extracted with 50 mL of hexane twice. Extracts were reduced in volume to ∼3 mL. Samples were fractionated using 1% water-deactivated silica columns with 50 mL of hexane, 50 mL of 40% dichloromethane in hexane, and 100% of dichloromethane. The 40 and 100% fractions, which contained the chlorinated compounds, were combined, solvent exchanged, and reduced in volume under a gentle stream of nitrogen to ∼200 µL. Samples were transferred to GC autoinjector vials with 500-µL inserts containing a known amount of 2,4,6-trichlorobiphenyl (PCB-30) (AccuStandard Inc.) as a recovery standard. Instrumental Analysis. The extracts were analyzed using a Hewlett-Packard gas chromatographic mass spectrometer (5973) operated in electron impact (EI) and electron capture, negative ionization (ECNI) modes. The instrument was equipped with a 60-m DB-5MS column (250-µm i.d., 0.25-µm film thickness; J&W Scientific, Folsom, CA). The injection port was maintained at 285 °C; 1 µL was injected in the pulsed injection, splitless mode with a 1.9-min vent time. Helium was the carrier gas at an average velocity of 25 cm/s. The temperature program began at 80 °C for 4860

Analytical Chemistry, Vol. 72, No. 20, October 15, 2000

1 min and was then ramped at 10 °C/min to 210 °C, 0.8 °C/min to 255 °C, and 15 °C/min to 300 °C, where the column was held for 10 min. The transfer line between the GC and the MS was heated to 280 °C. The ion source was operated at 230 and 150 °C in EI and ECNI modes, respectively, and the quadrupoles were operated at 150 and 106 °C in the EI and ECNI modes, respectively. Mass spectra for qualitative analyses were acquired using EI and ECNI by scanning from 100 to 500 Da. Data for quantitative analyses were acquired using a selected ion-monitoring program, which included ions at m/z 238, 272, 308, 310, 342, 344, 378, and 380. The reason for selecting these ions will be discussed below. The ions at m/z 308, 342, and 378 were used for quantitation, while the others were used for confirmation. The chlorinated compounds of interest were quantitated using relative response factors (RRFs) determined from a standard of three authentic compounds relative to p,p′-methoxychlor olefin. Recovery of the internal standard was determined using the RRF of methoxychlor olefin relative to PCB-30, the recovery standard. Kovats retention indexes (RIs) were determined by analyzing a mixture of n-alkanes ranging from n-octane (C8H10) to ntetracontane (C40H82) (AccuStandard) using the GC/MS scan method as described above (except the mass range was 50-500 Da). Quality Assurance. For both qualitative and quantitative analyses, a method blank, containing only the extraction solvents, was carried through the entire process. No chlorinated compounds were observed in the blanks. Internal standard recoveries in the quantitative extractions averaged 73 ( 17%. All solvents were spectroscopic grade. Silica was pre-extracted with dichloromethane, and the Na2SO4, alumina, and glass wool were heated in a muffle furnace at 450 °C for at least 4 h prior to use. RESULTS AND DISCUSSION The EI mass spectra from the 50 largest peaks in each gas chromatogram of each fraction from each paper sample extract were analyzed using the National Institute of Standards (NIST) mass spectral database. The database consistently matched one to four GC peaks in the 40% dichloromethane fractions with the known mass spectrum of methoxychlor olefin (see Figure 1, A); this compound is a degradation product of the pesticide methoxychlor (see Figure 1, B). Like the mass spectrum of methoxychlor olefin, all of the unknown mass spectra showed a molecular ion at m/z 308 with a 2-chlorine isotopic pattern. The ECNI mass spectra of the unknown compounds confirmed the molecular weight of 308 with two chlorines. We obtained standards of p,p′and o,p′-methoxychlor olefin (Accustandard Inc.), and we found that their GC retention times did not match any of our unknown compounds. Furthermore, the mass spectra of both methoxychlor olefin isomers showed a modestly abundant ion at m/z 195, which was not present in the mass spectra of our unknowns. On the basis of these discrepancies, we concluded that our unknown compounds were not methoxychlor olefins, but on the basis of the strong similarities in the mass spectra, we concluded that our unknown compounds were probably isomers of these olefins. A simple isomeric shift would be to move one ring to the other olefinic carbon. Thus, we hypothesized that our unknown compounds were 1,2-substituted ethylene isomers rather than 1,1substituted isomers. We searched the literature for compounds

Figure 2. Electron impact (EI, left) and electron capture, negative ionization (ECNI, right) mass spectra of D (top) and E (bottom). The EI and ECNI mass spectra of compounds C and D were identical.

of this type and with the molecular formula of C16H14Cl2O2, and we found that dichlorinated dimethoxystilbenes (see Figure 1, C-E) were occasionally cited as impurities in and degradation products of methoxychlor and also as byproducts formed during the production of bleached paper products.3-5 In fact, we were able to obtain authentic samples of compounds C-E (see Figure 1) from Bruce McKague at the University of Toronto.6 E had exactly the same retention index (RI ) 2536) and mass spectrum as one of the larger unknown GC peaks that we had found in the paper. C (RI ) 2301) also had a retention index similar to one of the larger unknown GC peaks (RI ) 2300), but the mass spectrum showed significant differences in ion abundances, indicating a different compound. The mass spectrum of E also matched that of three other GC peaks we had found in the paper extract. We also noticed other GC peaks that had mass spectra similar to that of E, but the molecular ions had been shifted by 34 and 68 Da, indicating the presence of three and four chlorines, respectively. Thus, we suspected that we had also found higher chlorinated homologues of these stilbenes. The unchlorinated dimethoxystilbenes are naturally occurring in wood and serve as (3) Mitchell, R. H.; West, P. R. Constituents of Commercial Methoxychlor (DMDT, 2.2 bis-(p-methoxyphenyl)-1,1,1-Trichloroethane). In Advances in Pesticide Science; Geissbuhler, H., Ed.; Pergamon Press: New York, 1979; pp 613-616. (4) West, P. R.; Chaudhary, S. K.; Branton, G. R.; Mitchell, R. H. J. Assoc. Off. Anal. Chem. 1982, 65, 1457-1470. (5) McKague, A. B.; Shen, X.; Reeve, D. W. A Comparison of Chlorinated Organic Material Produced by Chlorine and Chlorine Dioxide Bleaching of Kraft Pulp. In Chlorine and Chlorine Compounds in the Paper Industry; Turoski, V., Ed.; Ann Arbor Press: Chelsea, MI, 1998; pp 247-252. (6) McKague, A. B. University of Toronto, personal communication, 2000.

antifungal, antibacterial, and antifeeding agents.7-9 These compounds are also called pinosylvins and pinosylvin dimethyl ethers when hydroxy or methoxy functional groups are present in the 3 and 5 ring positions. For simplicity, we will refer to the methoxy substituted molecules as pinosylvins throughout this paper. The EI and ECNI mass spectra of D (the trans isomer) are shown in Figure 2A and B, respectively. The mass spectra of the cis and trans isomers (C and D) were the same under both ionization modes, so only one set of spectra are shown. The EI and ECNI spectra for E are shown in Figure 2C and D, respectively. The mass spectra of the two isomeric types show some major differences. The EI and ECNI mass spectra for the isomer in which the chlorines are substituted on the ethylene moiety (see Figure 2A and B) indicate that the molecular ion at m/z 308 is the most abundant ion under both ionization conditions. The EI spectrum of this isomer shows two successive losses of chlorine to give ions at m/z 273 for (M - Cl)+ and at m/z 238 for (M - Cl2)+. The ECNI spectrum shows a very abundant molecular ion at m/z 308 and a weak ion at m/z 273 representing (M Cl)-. In contrast, the EI and ECNI mass spectra of the compound in which the chlorines are substituted on the 2 and 6 positions of the ring (see Figure 2C and D) show that the molecular ion at m/z 308 is not the most abundant ion in the mass spectrum. Instead, in the EI spectrum, the ion at m/z 238 due to (M - Cl2)+ is the most abundant, and in the ECNI spectrum, the ion at m/z 273 due to (M - Cl)- is the most abundant. (7) Celimene, C. C.; Micales, J. A.; Ferge, L.; Young, R. A. Holzforshung 1999, 53, 491-497. (8) Ali, M. A.; Debnath, D. C. Bangladesh J. Sci. Ind. Res. 1997, 32, 20-24. (9) Zimmerling, T. N.; Zimmerling, L. M. J. Chem. Ecol. 1996, 22, 2123-2132.


4861

Table 1. Summary of GC Peaks Found in Various Bleached Paper Productsa. retention index

homologue largest m/z ng/g of paper


2 238 34

2 238 220

3 272 2

Microprint One (Georgia-Pacific) 3 2 2 272 238 238 b 14 500

3 272 2

3 272 6

Inkjet 24 (Union Camp) 2 238 12

3 272 3

3 272 1

Copy Plus (International Paper) 3 2 2 272 238 238 1 7 340

3 272 21

2 238 19

Primary Image (Xerox) 2 238 45


3 272 2

2 238 47

Coffee Filters (Kroger) 2 238 78


130 (60)

2 238 18

Paper Towels (Kimberly-Clark) 2 238 39


57 (68)

2 238 1

2 238 150

2568

770 (65)

2414

2 238 13

2543

3 272 2

2380

2 238 7

2536 (E)

total concn (ng/g) (% RI ) 2536)

2300


2523

2587

2260

4 306 4

4 306 b

32 (38) 3 272 2

530 (64)

3 272 1

68 (66)

aThe peaks are listed by their Kovats retention index. “Homologue” refers to the number of chlorines; the total concentration is the sum of the peaks listed. The name of the paper product is listed above each section with the manufacturer’s name in parentheses. b No quantitative data available.

These mass spectra indicate that the chlorines on the aromatic ring next to the methoxy groups are more easily lost from the molecule than those on the ethylene moiety under both EI and ECNI ionization conditions. This is most likely due to the electronwithdrawing effect of the methoxy groups on the aromatic ring. These shifts in relative ion abundances allow us to make predictions about the locations of the chlorines in the other congeners and homologues that we have found in the paper products. This is important because there are no known standards of chlorinated pinosylvins, other than the three described here. Six bleached and three nonbleached paper extracts were analyzed under both EI and ECNI conditions. Nine GC peaks were consistently found in two or more of the bleached paper samples with similar mass spectra. No chlorinated compounds were found in any of the nonbleached paper samples. The retention indexes and the mass spectra associated with these peaks are summarized in Table 1. Of C-E, only the isomer with the chlorines on the ring (E, RI ) 2536) was present in our paper samples. It was identified by its retention index and mass spectrum. On the basis of the high abundance of the (M - Cl2)+ ion relative to the molecular ion, all of the other dichlorinated compounds seem to have the chlorine atoms located on the aromatic ring. A representative EI mass spectrum (RI ) 2300) of one of the other dichlorinated compounds is given in Figure 3A. Note the abundant ion at m/z 238 (M - Cl2)+, and note how well the mass spectrum compares to that shown in Figure 2C. Of the four dichlorinated compounds reported in Table 1, none of their mass spectra showed the molecular ion as the dominant feature, indicating that none of these compounds had chlorine atoms on the ethylene moiety. This is contrary to results reported by McKague, who 4862 Analytical Chemistry, Vol. 72, No. 20, October 15, 2000

found that, of the two most abundant GC peaks in bleached paper pulp (D and E), one compound had the chlorines on the ethylene moiety.6 The trichlorinated pinosylvins were identified based on ions shifted by 34 Da and on a trichlorinated isotopic pattern at m/z 342 () 308 - H + Cl). The trichlorinated pinosylvins showed mass spectra similar to that of the dichlorinated compounds; that is, the molecular ion was never the most abundant. A typical EI mass spectrum for a trichlorinated isomer (RI ) 2543) is shown in Figure 3B. If two of the three chlorines had been on the ethylene moiety, we would have expected the molecular ion to be most abundant feature in the spectrum. Since it is not, we suspect that the three chlorines are on one or both of the rings. The possible substitution patterns will be discussed later in this paper. One tetrachlorinated congener (RI ) 2568) was identified in the paper samples under both EI and ECNI conditions. Like the trichlorinated homologue, the mass spectrum of this compound (see Figure 3C) is very much like that of the dichlorinated compounds except that the ions are shifted up by 68 Da. The molecular ion now is at m/z 376 () 308 - 2H + 2Cl) with a fourchlorine isotope pattern, and the major fragment ion is at m/z 306 for (M - Cl2)+ with a two-chlorine isotope patterns. As with the other homologues, the molecular ion is not the most abundant, indicating that the chlorines are not on the ethylene moiety. In this case, at least one of the four chlorines must be on the ring without the methoxy groups. With the exception of E, the positions of the chlorines on the rings of the other compounds are not known and cannot be deduced from their mass spectra. However, noticing a large

assumption we made based on the mass spectral data discussed earlier. Second, from the known ortho- and para-directing properties of the methoxy groups and from the unsubstituted ring’s link to the ethylene moiety, we chose only configurations in which two to four chlorine atoms were substituted on the 2, 2′, 4, 4′, 6, or 6′ carbons.10 Third, we assumed chlorination occurred simultaneously and not sequentially, negating the ortho- and paradirecting properties of chlorine. Fourth, for steric reasons, we assumed that only trans-substituted isomers were possible. Using these constraints, we calculated the dipole moments for 26 isomers substituted with two, three, and four chlorine atoms.

Figure 3. Representative electron impact mass spectra of di-, tri-, and tetrachlorinated pinosylvin dimethyl ethers extracted from bleached paper products.

variation in the retention indexes for compounds C-E (2301, 2218, and 2536, respectively), we thought that it might be possible to use these GC retention characteristics to identify the substitution patterns. To do this, we needed to relate the GC elution characteristics of these compounds to a basic physical property, such as polarity. Obviously, dipole moments were not known for these compounds, but with PC-based modeling software, these properties can be easily estimated. By correlating the calculated polarity of compounds C-E with their retention indexes, we could then estimate the retention indexes of other, hypothesized, compounds. Using PCMODEL software (Serena Software, Bloomington, IN), we constructed C-E, minimized the intramolecular electrostatic repulsion, and calculated the dipole moments of the minimized structures. The calculated dipole moments of compounds C-E are 2.5, 1.3, and 4.1 D, respectively. These are significant differences, which mirror the large difference in retention index; and this suggests that the GC elution order is dictated (at least in major part) by changes in dipole moment. Using these results we can predict the retention indexes of all other pinosylvin isomers with two, three, and four chlorines, but to restrict the number of isomers we had to investigate, we implemented four chemical constraints: First, we assumed that there would never be chlorine atoms on the ethylene moiety, an

On the basis of the work of Ong and Hites, it was expected that a correlation between retention index and the dipole moment squared would be linear.11 For C-E, this correlation gave a slope of 21.246 and an intercept of 2176.4 with a correlation coefficient (r2) of 0.9981. From this relationship, we estimated the RI values of the eight dichlorinated compounds. In addition, we noticed a consistent shift of 154 RI units between the GC peaks at 2260, 2414, and 2568, compounds with two, three, and four chlorine atoms, respectively. Thus, to estimate the RI of the tri- and tetrachloro compounds, we used the dipole moment squared-RI relationship for the dichloro compounds but added 154 or 308 RI units to the intercept. Equations 1-3 show these relationships,

RI (dichlorinated) ) 21.246D2 + 2176.4 2

RI (trichlorinated) ) 21.246D + 2330.4 2

RI (tetrachlorinated) ) 21.246D + 2484.4

(1) (2) (3)

where D represents the calculated dipole moment. The RI results for the restricted list of 26 compounds are given in Table 2. Within each homologue, the structures are arranged by increasing estimated RI value. We recognize, of course, that these RI values have considerable error; therefore, as we compare them to the experimental RI values (see Table 1), we are looking for only an approximate agreement of (70 RI units. This confidence limit was based on the error of the dipole moment squared-RI regression. The first unknown compound at RI 2260 was dichlorinated, and its experimental RI value agrees with the estimated values for the 4,4′- and 2′,4′-substituted isomers (2212 and 2245, respectively). The second unknown dichlorinated compound at RI 2300 shows agreement with the 2′,4′- and 2′,6′-substituted isomers (2245 and 2368, respectively). The third unknown dichlorinated compound at RI 2523 has a retention index that agrees with only the (10) Ege, S. Organic Chemistry, 2nd ed.; D. C. Heath and Co.: Lexington, MA, 1989. (11) Ong, V. S.; Hites, R. A. Anal. Chem. 1991, 63, 2829-2834.


4863

Table 2. Estimated Dipole Moments (in debyes) for Selected Di-, Tri-, and Tetrachlorinated Pinosylvin Dimethyl Ether Isomers and the Assignment of the Unknown GC Peaks. isomer

dipole moment

est RI

assignment

4,4′ 2′,4′ 2′,6′ 2,4 2,2′ 2,6 2′,4 2,4′ 2,4,6 2′,4′,6′ 2,4,4′ 2′,4,4′ 2,2′,6′ 2,2′,6 2,2′,4 2,2′,4′ 2′,4,6′ 2,4′,6 2,2′,4,6 2,4,4′,6 2,2′,6,6′ 2,2′,4,4′ 2,2′,4′,6′ 2′,4,4′,6′ 2,2′,4,6′ 2,2′,4′,6

1.3 1.8 3.0 3.1 3.5 4.1 4.4 4.5 0.2 0.9 2.6 2.7 3.1 3.6 4.0 4.3 5.1 6.2 1.8 2.2 2.4 3.1 3.4 3.4 4.4 5.6

2212 2245 2368 2381 2437 2534 2588 2607 2331 2348 2474 2485 2535 2606 2670 2723 2883 3147 2553 2587 2607 2689 2730 2730 2896 3151

2260a 2260 or 2300 2300

a

2536 (E) 2523 2380 2380 or 2414 2414a or 2543 2543 2543 or 2587 2543 or 2587

2568 2568a 2568

Selected on the basis of the consistent 4,4′ substitution pattern.

estimated RI value for the 2′,4-substituted isomer (2588). The trichlorinated compound at RI 2380 has RI values that agree with those of the 2,4,6- and 2′,4′,6′-substituted isomers (2331 and 2348, respectively). The next trichlorinated compound at RI 2414 has an RI value that agrees with those of the 2′,4′,6′- and 2,4,4′substituted isomers (2348 and 2474, respectively). The third unknown trichlorinated compound (RI ) 2543) could be one of four isomers: 2,4,4′, 2′,4,4′-, 2,2′,6′-, or 2,2′,6-substituted (with estimated RI values of 2474, 2485, 2535, and 2606, respectively). The last trichlorinated unknown compound at RI 2587 could be either the 2,2′,6′- or 2,2′,6-substituted isomer. The one tetrachlorinated compound has a RI of 2568, and this agrees with the estimated values of the 2,2′,4,6-, 2,4,4′,6-, and 2,2′,6,6′-substituted isomers (2553, 2587, and 2607, respectively). The regular shift of 154 RI units between the peaks at 2260 (2 Cl), 2414 (3 Cl), and 2568 (4 Cl) suggests that these compounds differ from one another only by the addition of a chlorine. Examining the possible substitution patterns for these three peaks, we notice that substitution on the 4 and 4′ positions is a common feature. Thus, we suggest that the peak at RI 2260 is the 4,4′ isomer, the peak at RI 2414 is the 2,4,4′ isomer, and the peak at 2568 is the 2,4,4′,6 isomer. This regular addition of a chlorine, first on the 2 position and then on the 6 position, would likely cause the regular shift in retention index. These GC peak assignments are summarized in the last column of Table 2. In this way, we are able to make reasonable predictions about the structures of the compounds we found in the paper products. We should note that this analysis was not intended to be an indepth modeling exercise. We simply took a common sense approach to predicting logical structures using basic organic 4864


chemistry knowledge and simple modeling techniques for compounds for which there were no known standards. We have included only GC peaks for which we were able to obtain mass spectra in both EI and ECNI modes. There is evidence for other isomers in each homologue group. These other peaks are often tetrachlorinated homologues. They were observed in ECNI mode but not in EI mode, probably due to the increased electron capture efficiency of multiply halogenated compounds in ECNI mode. Thus, the nine peaks described in Table 1 are not a complete list of chlorinated pinosylvin compounds present in bleached paper products. Armed with these qualitative identifications, we then extracted a new set of paper samples for quantitative analysis. Recoverycorrected concentrations of total chlorinated pinosylvins range from 32 to 770 ng/g based on the weight of the paper product, these concentration data are presented in the right-most column of Table 1. Quantitative data were obtained under EI SIM conditions, which provided greater sensitivity than the EI scan mode used for qualitative analysis. Therefore, peaks that were quantitated included those described in Table 1, as well as a few others. In all cases, most of the total mass was a result of two dichlorinated compounds: one was E (the 2,6 isomer), and the other was the GC peak with an RI of 2300, which is likely the 2′,4′ or 2′,6′ isomer. The percent contribution of E to the total mass is also included in Table 1. It is interesting that, with the exception of the Union Camp paper, the percent contribution from E is consistently between 60 and 68%. The same isomer only constitutes 38% of the total chlorinated pinosylvins found in the Union Camp paper, which also has the lowest quantity of total chlorinated pinosylvins. A typical piece of sheet paper used in this study weighed ∼4 g; thus, there are 2-3 µg of chlorinated pinosylvins per sheet of paper for the Georgia-Pacific and International Paper products. The coffee filters weighed ∼1 g each, which would give 100 ng/filter. The paper towels were rolled, so no such comparison can be made. The toxicity of the compounds reported here is not known. However, the toxicity of isomers and structural analogues of these dichlorinated compounds has been investigated. These compounds include methoxychlor olefin, a degradation product of methoxychlor, and other isomers that are byproducts and are present in the technical methoxychlor mixture. These compounds are reported to be proestrogenic.3,4 Increased enzymatic activity was seen in fish exposed to a hydroxylated trichlorinated pinosylvin compound, which had been isolated from bleached Kraft mill effluent.12 Finally we note that these pinosylvins are structurally analogous to the synthetic hormone, diethylstilbesterol (DES; see Figure 1, F). ACKNOWLEDGMENT We thank Bruce McKague at the Pulp and Paper Center, University of Toronto for helpful discussions and for providing the dichloropinosylvin standards. We also thank Joe Gajewski at Indiana University for assistance with the modeling and prediction of structures. Received for review April 26, 2000. Accepted August 3, 2000. AC000474G (12) Burnison, B. K.; Comba, M. E.; Carey, J. H.; Parrott, J.; Sherry, J. P. Environ. Toxicol. Chem. 1999, 18, 1882-2887.

Identification of Chlorinated Dimethoxystilbene Isomers and

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