Dichloromyristic Acid, a Major Component of Organochlorine Load in

Environ. Sci. Technol. 1997, 31, 535-541

Dichloromyristic Acid, a Major Component of Organochlorine Load in Lobster Digestive Gland† JOYCE E. MILLEY, ROBERT K. BOYD, AND JONATHAN M. CURTIS* Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1 CHARLES MUSIAL Charles Musial Consulting Chemist, P.O. Box 949, Monticello, Mississippi 39654 JOHN F. UTHE Marine Environmental Sciences, Department of Fisheries and Oceans, P.O. Box 550, Halifax, Nova Scotia, Canada B3J 2S7

It is well established that PCBs and chlorinated pesticides account for only some 15% of extractable organically-bound chlorine (EOCl) in samples from marine and non-marine environments. Work by other investigators on marine sediments and lipids from highly contaminated fish, collected near kraft pulp mills, has shown that chlorinated alkanoic acids contribute significantly to the EOCl. The present investigation extends this work to lipids from lobsters (Homarus americanus) captured in an industrial harbor well removed from any pulp mill effluent. The relatively low chlorine content of these lipids (30100 µg g-1) necessitated development of fractionation and analysis procedures more discriminating and sensitive than those used previously. Neutron activation analysis for total chlorine was used to monitor the extraction, cleanup, transesterification, and selective EOCl enrichment of the lipids. Fractionation on a Sephadex LH-20 column then concentrated the EOCl into fractions separated from the bulk of the lipid. Mass spectrometric detection using dissociative electron capture, monitoring only chloride ions, identified those GC peaks containing chlorine. Conventional negative ion mass spectrometry provided mass spectra for peaks of interest and enabled identification of a dichloromyristic acid as a lipid component accounting for ca. 20% of the EOCl on a semi-quantitative basis.

Introduction The total organochlorine contents of water, sediments, and biological tissues from marine and non-marine environments exceed those accounted for by common contaminants such as polychlorinated biphenyls (PCBs) and chlorinated pesticides (1-10). More hydrophilic unidentified organochlorines are believed (11) to originate from humic material. Enzymatic halogenation by inorganic halides plus haloperoxidases is a well-established phenomenon (12, 13), and many chlorinated natural compounds are known (14-16). Concentrations of natural chlorinated aromatics in soils associated with the occurrence of basidiomycetes (higher fungi) can exceed (16) * To whom correspondence should be addressed. Telephone: (902)-426-5680; fax: (902)-426-9413; e-mail: [email protected]. † NRCC No. 39735.

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hazardous-waste norms applied to analogous anthropogenic chlorophenols. Here, extractable organically-bound chlorine (EOCl) refers to chlorinated compounds extracted by non-polar solvents and subjected to a Folch wash followed by a solvent change (see below). Early work (17-19) on EOCl in fish used neutron activation analysis (NAA) to track the chlorine through simple fractionation schemes. Extractable organically-bound bromine (EOBr) in marine fish chromatographed with triacylglycerols and sterol esters (20), and it was suggested (21) in 1980 that 60-80% of the EOBr from marine fish was present as brominated fatty acids. Remberger et al. (22, 23) analyzed EOCl fractions from marine sediments collected from sites close to kraft pulp mill effluents. Measurement was by GC with flame ionization detection (FID), and qualitative identification was by GC/ MS. Free chlorinated acids in these fractions were dominated (22, 23) by chlorinated alkanoic acids and chlorinated resin acids, identified previously in mill effluents (24, 25). Transesterification yielded additional amounts of chlorinated alkanoic acids (as methyl esters). With the exception of the PCBs and pesticides, all compounds identified in the sediments were present in the contaminating pulp bleach effluents (23), and it has been proposed (26, 27) that sediments can be a source of EOCl contamination in fish lipids. A proposal (28) that some of the unidentified EOCl in fish lipids might be chlorinated fatty acids has been pursued experimentally by Weseń et al. (29-36). NAA was used to track the chlorine content through the extraction and fractionation procedures, together with analyses of chlorinated fatty acid methyl esters (FAMEs) using GC with an electrolytic conductivity detector (ELCD) operated in the halogen-specific mode (37), which afforded (31) a detection limit of 0.25 ng. Mass spectrometric detection (30) using selected ion monitoring (SIR) gave instrumental detection limits for model compounds (e.g., methyl dichlorostearate) of 0.1 ng for both ammonia positiveion chemical ionization (PICI-MS) of FAMEs and for negativeion chemical ionization (NICI-MS) of the pentafluorobenzyl esters. Detection limits for GC using a FID or electron capture detector (ECD) gave detection limits (30) for dichlorinated FAMEs of 1 and 0.1 ng, respectively. These instrumental detection limits do not apply to analyses of complex lipid extracts containing low levels of the chlorinated compound. For fish lipid extracts containing 30-100 µg Cl g-1, detection of trace EOCl components using available GC/MS techniques is not possible after cleanup by simple methods such as liquid-liquid partitioning (29, 31, 32). However, eels from a fiord contaminated by effluent from a chlorine-bleach sulfite-based pulp mill provided Weseń et al. (31-33, 35, 36) with lipid containing 1200 µg Cl g-1. This enabled identification of dichlorostearic acid (both threo and erythro) in the lipid, using both GC/ELCD (31) and GC/MS with both electron ionization (EI) and PICI (32, 33, 36). In order to obtain meaningful mass spectra, the chlorinated FAMEs were preconcentrated. In one approach (36), silver ion complexation was used to deplete unsaturated FAMEs, followed by selective removal of straight-chain compounds by urea co-crystallization to leave the branched-chain (including chlorinated) FAMEs in solution. A different enrichment method (33) used cryogenic trapping of effluent from preparative GC/ELCD experiments prior to GC/MS analysis. Other work (35) on the same eel lipids has indicated the presence of diastereomers of tetrachlorostearic acid and probably also of various isomers of dichlorinated, monounsaturated C18, C16, and C14 fatty acids. Weseń et al. also studied lipids from herring (32) and shellfish (34). Some mussels (34) provided lipid extracts with

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chlorine loads from 50 to 790 µg g-1 and yielded a correlation between total chlorine levels and the distribution of homologous chlorinated fatty acids. At higher chlorine levels dichlorostearic acid dominated, but in specimens collected further from the pollution source, the chlorine levels dropped, and the chlorinated fatty acids were mostly dichloromyristic acid with some dichlorostearic acid. In control specimens with chlorine levels in the lipid of ca. 50 µg g-1, dichloromyristic acid dominated the profile (34). This pattern (34) may reflect in some way the findings of Conacher et al. (38) on the β-oxidative metabolism of dichlorostearic acid in rats, which resulted in chain shortening to dichloromyristic acid but no further. In related work, McKague and Reeve (39) claimed to have identified chlorination products of linoleic acid in chlorinated wood pulp. Also, bacteria and fungi have been discovered (40-42) that incorporate chloroalkanes into ω-chloro fatty acids. The present work was devoted to the investigation of chlorinated compounds in lipid extracts of digestive glands of American lobster (Homarus americanus), captured in an industrial harbor near an untreated municipal sewage outflow far removed from the nearest pulp mill. The total chlorine content of the lipid was more than an order of magnitude lower than that in the eel lipids investigated by Weseń et al., and it was necessary to develop additional experimental approaches.

Experimental Section Samples. Intact digestive glands were taken from lobsters (H. americanus) captured in 1992 and 1995 near an untreated municipal sewage outflow in an industrial harbour, 70 mi from the nearest pulp mill that was converted to a thermomechanical process in the mid-1980s. Apart from PCBs and pesticides, the only known sources of chlorine were inorganic chloride and constituents of the sewage, including domestic bleach. The digestive glands were pooled, homogenized, and sealed in glass ampules under argon, all within ca. 4 h of sampling. Extraction, Cleanup, and Fractionation. Many procedures used in this work followed literature methods (9, 2936), and only significant extensions are described here. Because of the relatively low levels of chlorine in these samples, meticulous precautions were necessary. Full details of these procedures are given in a technical report (43) available from the authors. Folch Wash of Extracted Lipid. The tissue homogenate was extracted with acetone using an ultrasonic probe. A hexane-toluene mixture was then added, followed by further ultrasound treatment. Prior to NAA, protein and inorganic chloride were washed from the lipid extract using the Folch solvent system [chloroform-methanol-water (44), but not doped with inorganic salts since these were potential sources of chloride]. The washed lipid was exchanged with hexane at least three times to remove all traces of chloroform. Preconcentration of Chlorinated Fatty Acids. Following transesterification to FAMEs, the same strategy as that of Mu et al. (36) was used to preconcentrate the chlorinated FAMEs. Removal of unsaturated FAMEs by silver ion complexation used the procedure of Peers and Coxon (45) as also reported by Mu et al. (36). However, due to disappointing chlorine enrichments and to fears of contamination, this step was omitted in the later work reported here (Scheme 1). Urea inclusion complexation to selectively remove straight-chain FAMEs from solution initially used the urea column technique of Cason et al. (46), but problems with channel formation in the column led to adoption of the urea co-crystallization method of Wijesundera and Ackman (47). Fractionation of Lipid Extracts. In the present work, the lipid extract prior to transesterification was fractionated into lipid classes as described by Hakansson et al. (9) using silicagel chromatography. However, concentrations of orga-

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SCHEME 1. Flow Chart for Cleanup, Preconcentration, and Fractionation Procedures Used for Lipid Extract from 1995 Lobster Samplea

a

Details are in the text and in ref 43.

nochlorines in the resulting fractions were too low to permit successful GC/MS analysis. Accordingly, a different strategy (Scheme 1) was adopted in which transesterification and urea co-crystallization were followed by fractionation on a column of Sephadex LH-20, prepared by pre-swelling 40 g of Sephadex LH-20 chromatography packing (Pharmacia Biotech AB, Uppsala, Sweden) in methanol for several days, transferring to a glass column (100 × 1.5 cm), washing with methanol, and allowing it to settle overnight. The transesterified lipid (500 mg) from the urea co-crystallization supernatant was dissolved in a minimum quantity of methanol and transferred quantitatively to the column. Fractions were eluted with methanol and collected in acid-washed, solvent-rinsed, preweighed test tubes using a FOXY II fraction collector (ISCO Inc. Lincoln, NE) set to collect 100 drops per tube with approximately 3-4 s between drops. A small aliquot of each fraction was spotted on a silica-gel TLC plate, immediately sprayed with p-anisaldehyde, and heated to visualize which fractions contained significant quantities of material. Each selected fraction was taken to approximate dryness under a nitrogen stream, then dried to constant weight under active vacuum, dissolved in hexane, and made up to volume so as to not exceed a total concentration of 1-2 mg mL-1. These fractions accounted for 98.3% of the mass of lipid applied to the column. Synthesis of Chlorinated Fatty Acid Standards. Monoand dichlorinated fatty acids were prepared from unsaturated precursors: oleic (18:1, cis ∆-9), elaidic (18:1, trans ∆-9), vaccenic (18:1, both cis and trans ∆-11), palmitoleic (16:1, cis ∆-9), and myristoleic (14:1, cis ∆-9). Standard synthetic procedures were used. Chlorination of an unhindered double bond proceeds in an anti fashion (48), so that cis and trans unsaturated fatty acids should yield the corresponding threo and erythro dichlorinated saturated acids, respectively. Structures and purities were checked by NMR and by GC/MS of the methyl esters, as described elsewhere (43). GC/ECD and GC/MS Analyses. GC/MS experiments used an AutoSpec OA-TOF hybrid mass spectrometer (Micromass, Manchester, U.K., only the double-focusing analyzer was used here), interfaced to a HP 5890 Series II GC with cool oncolumn injector (Hewlett-Packard, Palo Alto, CA). A 30-m DB-5 capillary column (0.25 mm i.d., 0.25 µm film thickness) and a 30-m DB-WAX column (0.25 mm i.d., 0.25 µm film thickness) both from J&W Scientific (Folsom, CA) were used. For NICI with methane, two sets of conditions were used. In one case, the objective was to achieve “hard” ionization, viz., dissociative electron attachment (DEA) to produce predomi-

FIGURE 1. Total ion chromatograms (m/z 100-600) from GC/MS analyses with EI of FAME mixtures; 30-m DB-5 column. (a) FAMEs from transesterification of pooled fractions 2 and 3 from silica-gel chromatography of the lipid fraction. (b) FAMEs in the urea co-crystallization supernatant of pooled fractions 2 and 3 following silver ion complexation. (c) Dichloro-FAME standards. Note that no dichloro-FAMEs were detected in panels a and b (see text). nantly chloride ion from organochlorine components, so that SIR of m/z 35 and 37 indicated those GC peaks containing chlorine. The best conditions for DEA involved an ion source temperature as high as possible (250 °C) without excessive contamination (sooting) of the source, electron acceleration potential 250 eV, total electron emission 2 mA, and a methane pressure of 1-3 × 105 mbar as indicated on the source housing ion gauge. A similar approach has been described previously (49). To obtain information on molecular mass and structure, “soft” ionization conditions as described by Hites et al. (5052) involved a source temperature of 180 °C, electron accelerating potential of 40-50 eV, total emission current of 2 mA, and a higher source pressure. Under soft ionization conditions, the mass spectrometer was scanned over the range m/z 550-25 at 0.8 s per decade with a 0.2-s interscan delay. Other details of GC/MS operating conditions are given elsewhere (43). GC/ECD analyses used an HP 5890 Series II GC and a 30-m HP5-MS capillary column (Hewlett-Packard, 0.25 mm i.d., 0.25 µm film thickness).

Results and Discussion Total EOCl in Extract of Lobster Digestive Gland. A correction for a paraffin oil blank, typically less than 1 µg of Cl/g of lipid, was applied to all NAA measurements. The reproducibility, based on extractions performed 6 months apart, was better than 15%. The total chlorine content determined in the 1995 sample extract (98 ( 13 µg of Cl/g of lipid, n ) 7) was about three times higher than in the 1992 sample (33 ( 2 µg of Cl g-1, n ) 3). The extracted lipid accounted for 25-30% of the drained wet tissue mass that was subsequently homogenized. The 1992 sample was analyzed for anthropogenic organochlorines, as described by Newsome et al. (10, 53). PCBs accounted for 3.5 µg of Cl g-1, and organochlorine pesticide residues (principally p,p′-

DDD and p,p′-DDE) accounted for 0.3 µg of Cl g-1, for a total of 3.8 µg of Cl g-1, i.e., ca. 12% of the total chlorine present as EOCl (54), in general agreement with previous findings (1-10). Fractionation of the 1992 Lipid Extract by Silica-Gel Chromatography. The distribution of chlorine among the four fractions from silica-gel chromatography (9, 43) of the 1992 sample extract was determined by NAA. Fraction 1 contained non-polar compounds including PCBs and chlorinated pesticides and accounted for about 20% of the total chlorine, reasonably consistent with the identification of 12% of the total chlorine as pesticides and PCBs and with previous findings (9). Fractions 2 and 3 (free fatty acids plus neutral acylglycerols) together accounted for 78% of the total chlorine in the extract. Although fraction 2 contained almost as much chlorine as fraction 3, the concentration of chlorine (µg of Cl/g of material in that fraction) was ca. 5 times higher in fraction 3. A dichlorostearic acid standard eluted in fraction 3. As noted previously (9) this fractionation method resulted in leaching of extraneous chlorine from the silica gel into the most polar fraction, leading (43) to irreproducible NAA data for fraction 4. Fractions 2 and 3 were pooled and transesterified. Similarly to Weseń et al. (36), the FAME mixture was subjected to silver ion complexation (45) to remove unsaturated FAMEs, thus increasing the chlorine concentration (µg of Cl/g of remaining lipid material, measured by NAA) by only some 12%. Urea co-crystallization (46), however, concentrated (ca. 3 times) the chlorine content into the supernatant. Analysis of this supernatant was done initially by GC/MS with EI, and Figure 1 compares total ion chromatograms (TICs) for retention times appropriate to C14-C24 FAMEs. Figure 1a shows the result for the FAMEs mixture obtained by transesterification of the pooled fractions 2 and 3, while Figure 1b


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FIGURE 2. Distribution of transesterified lipids from the urea cocrystallization supernatant (Scheme 1) among the LH-20 fractions. Black bars: masses of material in fractions (mg). White bars: masses (µg) of chlorinated compound(s) with GC characteristics similar to those of methyl dichloropalmitate (Figures 5 and 6), estimated by external calibration. Shaded bar: mass (µg) of methyl dichloromyristate estimated by external calibration. shows the TIC obtained for the urea crystallization supernatant. These two TICs are appreciably different from one another, particularly peaks A-D in Figure 1b, which are barely apparent in Figure 1a. Thus, although silver ion complexation removed highly unsaturated FAMEs (e.g., 22:6), other unsaturated FAMEs were removed to a lesser degree (e.g., about 20% of each of 22:4 and 18:3 were removed). The retention time of each of peaks A-D approximately matches that of one of the dichloro-FAME C16-C22 standards (Figure 1c). However, mass spectra obtained at the crests of peaks A-D bore no resemblance to those of the dichloro-FAME standards. Library searching suggested methyl esters of oxygenated fatty acids, but there were no satisfactory matches with any of the particular compounds of this type in the library. Additional GC/MS analysis of the enriched fractions using conventional soft NICI failed to identify any organochlorines. Thus the procedures of Weseń et al. (36), developed for their eel lipid, were insufficient for the 1992 lobster lipid with its substantially lower (factor of 35-40) chlorine content, even with the added cleanup provided by silica-gel chromatography (9). Fractionation of the 1995 Sample using Sephadex LH20. The 1995 lipid extract was transesterified immediately following the Folch wash (44) and subjected to urea cocrystallization but not to silver ion complexation and then fractionated on Sephadex LH-20 (Scheme 1). The chlorine content was monitored by NAA for all steps prior to the LH20 separation. After the Folch wash, the chlorine concentration was 98 µg/g of remaining lipid, but only 48 µg g-1 after transesterification, an uncontrolled loss of ca. 50%. Following the urea crystallization step, the co-crystallizing fraction contained 6 µg of Cl g-1, while the supernatant contained 75 µg of Cl g-1. Because irradiated NAA samples cannot be recovered for GC/MS analysis, the masses of the LH-20 fractions were used as the initial screening parameter. Fractions 40-70 contained most of the lipid (Figure 2, PCBs and chlorinated pesticides eluted in fractions 75-100, not shown). Figure 3 compares chromatograms obtained for fraction 60 using GC/ECD, GC/

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FIGURE 3. GC analyses (DB-5 column) of the LH-20 fraction 60. (a) Electron capture detector. (b) TIC from MS with EI; peaks are identified where possible (FAME identities in square brackets). (c) Chloridespecific GC/MS analysis using DEA conditions and monitoring only m/z 35 and 37. MS with EI, and GC/MS with DEA-NICI monitoring only m/z 35 and 37. The ECD chromatogram (Figure 3a) and the TIC from the GC/EIMS analysis (Figure 3b) yielded no useful information concerning the organochlorine compounds. The most intense peak in the chloride-specific chromatogram (Figure 3c) appeared at the retention time of the methyl 9,10-dichloromyristate standard. Figure 4b shows the relevant retention range of Figure 3c, for comparison with the DEA chromatogram for the dichloro-FAME standards (Figure 4a). In addition to the retention time match for methyl dichloromyristate, there is also a good match of a minor peak in Figure 4b with that of methyl dichloropalmitate (Figure 4a). In contrast, Figure 4c shows the TIC obtained also by GC/MS analysis of fraction 60, but using soft NICI conditions. The GC peak that matches methyl dichloromyristate is now a relatively minor feature, while that matching methyl dichloropalmitate is unobservable. A GC peak matching methyl dichloropalmitate was the major component in the DEA GC/ MS chromatograms (not shown) obtained for fractions 6164. The DEA-NICI GC/MS chromatograms (Figures 3c and 4b) are specific for chlorine-containing components, but the bulk of the material in fraction 60 is better characterized by the chromatograms obtained using GC/MS with EI (Figure 3b) and with full-scan soft NICI (Figure 4c). Each chromatogram in Figure 3 shows some badly fronting GC peaks, indicating overload of the GC column as also evidenced by the distorted baseline in the early stages of the DEA-NICI chromatogram (Figure 3c). The results shown represent a compromise between GC performance (the peaks in the DEANICI chromatograms are reasonably sharp) and absolute signal level. These analyses were repeated using a more polar GC column (DB-WAX, see Figure 5). The peak in Figure 4b at 12

FIGURE 4. GC/MS analyses (DB-5 column) of lipid samples. (a) Chloride-specific analysis of a mix of dichloro-FAMEs standards, using hard NICI conditions (DEA) and monitoring only m/z 35 and 37. (b) Chloride-specific analysis of LH-20 fraction 60, using DEA conditions and monitoring only m/z 35 and 37, expanded range of Figure 3c. (c) TIC from analysis of LH-20 fraction 60, using soft NICI conditions for full-scan mass spectra.

FIGURE 5. GC/MS analyses (DB-WAX column) of lipid samples, using soft NICI conditions and scanning the range m/z 550-25. (a) Reconstructed ion chromatogram (sum of intensities of m/z 35 and 37) obtained for a mix of dichloro-FAMEs standards. (b) Reconstructed ion chromatogram (sum of intensities of m/z 35 and 37) obtained for LH-20 fraction 60. (c) TIC from analysis of LH-20 fraction 60.


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FIGURE 6. NICI mass spectra obtained in GC/MS analyses using soft ionization conditions. (a) Constituent of LH-20 fraction 60, eluting at 21 min 41 s (Figure 5b). (b) Standard sample of methyl dichloromyristate at a low partial pressure in the NICI ion source. (c) Standard sample of methyl dichloromyristate at a high partial pressure in the NICI ion source. min 26 s, suggested to be methyl dichloromyristate, now appears (Figure 5b) at 21 min 41 s and still matches the retention time of the standard (Figure 5a). Moreover the NICI mass spectrum recorded at 21 min 41 s (Figure 5c) is shown in Figure 6a and is an excellent match for the corresponding spectrum obtained for the standard (Figure 6b). The fragment ion at m/z 239 corresponds to expulsion from the M•- ion of the elements of (H, 2Cl), and that at m/z 207 corresponds to further loss of the elements of methanol. A similar spectral match of the unknown to the standard was found for the data obtained using a DB-5 column (Figure 4, spectrum not shown), although an additional ion at m/z 262 arising from a partially co-eluting component could not be completely eliminated from the spectrum by background subtraction. The identification of this major chlorinated component of fraction 60 as a methyl dichloromyristate is thus confirmed. It was found that the soft NICI mass spectra of dichlorinated FAMEs were sensitive to the amount of material injected, as illustrated in Figure 6c, which shows a soft NICI spectrum of methyl dichloromyristate obtained using a higher partial pressure of sample in the ion source and resulting in a dramatic change in the high mass region of the spectrum. The isotopic cluster at m/z 345 and above is characteristic of the chloride attachment ion (M + Cl)- for this compound. Standards of 9,10- and 11,12-dichlorostearic acid (both threo and erythro) were available, and GC/MS analysis of their methyl esters showed that, although threo and erythro forms were separable by GC, positional isomers (chlorine substitution) were not. This negative result for dichloro-FAMEs agrees with previous work (55), which showed that the GC retention characteristics of monochlorinated FAMEs were almost entirely insensitive to the position of chlorination. Therefore, no information could be obtained on the positional isomerism of the dichloromyristic acid identified here, although the GC

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retention characteristics of its methyl ester were identical to those of the threo-9,10-dichloro C14 FAME standard. Unfortunately, no trans tetradecenoic acid precursors were available, so it was not possible to check that the threo and erythro C14 dichloro-FAMEs were indeed separable under the GC conditions used. The peak at 15 min 42 s in Figure 4b (DB-5 column) shifted to 28 min 22 s in Figure 5b (DB-WAX), which does not match the retention time of the methyl dichloropalmitate standard (Figure 5a). Moreover, the mass spectrum of this unknown compound (not shown) contained only the chloride ion under the same soft NICI conditions, which yielded an informative mass spectrum of the standard. This same unknown compound, or possibly a series of related compounds all with indistinguishable GC retention behavior, was distributed through LH-20 fractions 60-66 as shown in Figure 2, while in contrast methyl dichloromyristate was concentrated in fraction 60. The mass estimates for this unknown chlorinated compound (Figure 2) are based upon GC peak areas for chloride ion in DEA GC/MS experiments, using external calibration with a methyl dichloropalmitate standard, and are semiquantitative only. Fraction 60 (8.2 mg of total mass, 1.6% of the transesterified lipid) contained 77 µg of methyl dichloromyristate, based on similar external calibrations of GC/MS peak areas. Extrapolation of this result to estimate the concentration in the lipid extract itself is unreliable because transesterification resulted in a major loss (ca. 50%) of chlorine. However, by assuming that the fractional loss of total chlorine was the same as that of dichloromyristate, an estimate of 44 µg of dichloromyristic acid/g of lipid prior to transesterification, equivalent to ca. 20% of the total EOCl, can be made. No evidence for monochlorinated FAMEs was found in the present work thus far, in agreement with Weseń et al.

(29-36). Low-level contamination by a dichlorodiphenylsulfone was observed in all LH-20 fractions. Blank experiments showed that this contaminant was an artifact of the fractionation procedure.

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Acknowledgments

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We are indebted to Dr. M. Bewers of the Department of Fisheries and Oceans (Canada) for financial support for this work. Drs. P. Andrews, H. Conacher, F. Iverson, and H. Newsome of Health Canada provided helpful advice and information. Prof. R. Ackman of the Technical University of Nova Scotia provided assistance with lipid analyses. Analyses for PCBs and chlorinated pesticides were performed by Ms. D. LeBlanc. Mr. K. Chan performed the GC/ECD analyses. Dr. S. Ayer assisted with synthesis of chlorinated fatty acids, and NMR spectra were obtained by Dr. J. Walter and Mr. I. Burton. Dr. N. Ross provided protein analyses of the lipid extract. The NAA experiments were conducted by Dr. J. Holzbecher and Dr. A. Chatt of the Dalhousie University SLOWPOKE-2 Reactor Centre, under contract to the National Research Council of Canada. The helpful comments of two anonymous referees are acknowledged.

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(43) (44) (45) (46) (47) (48) (49) (50)

(51) (52) (53) (54) (55)

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Received for review May 8, 1996. Revised manuscript received September 24, 1996. Accepted September 27, 1996.X ES960407X X

Abstract published in Advance ACS Abstracts, December 15, 1996.


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Dichloromyristic Acid, a Major Component of Organochlorine Load in

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