Environ. Sci. Technol. 2004, 38, 1992-1997
Radiocarbon Evidence for a Naturally Produced, Bioaccumulating Halogenated Organic Compound
from discrete locations need to be analyzed before a clear understanding of the source (or sources) of this compound (and other unknown HOCs) is fully determined.
Introduction C H R I S T O P H E R M . R E D D Y , * ,† L I X U , † GREGORY W. O’NEIL,† ROBERT K. NELSON,† TIMOTHY I. EGLINTON,† D. JOHN FAULKNER,‡ ROSS NORSTROM,§ PETER S. ROSS,| AND S H E R Y L A . T I T T L E M I E R ⊥,∇ Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093, Environment Canada, Canadian Wildlife Service, National Wildlife Research Centre, Hull, Quebec, Canada K1A 0H3, Institute of Ocean Sciences, Fisheries and Oceans Canada, Sidney, British Columbia, Canada V8L 4B2, and Centre for Analytical and Environmental Chemistry, Carleton University, Ontario, Canada K1S 5B6
Halogenated organic compounds (HOCs) such as 1,1′dimethyl-3,3′,4,4′-tetrabromo-5,5′-dichloro-2,2′-bipyrrole (DBPBr4Cl2) and heptachloro-1′-methyl-1,2′-bipyrrole (Q1) have been detected worldwide, sometimes at high levels in Antarctic air, seabird eggs, the blubber of marine mammals, and, most notably, even human milk. To date, it has been difficult to determine whether these compounds are natural products or derived from industrial synthesis. Molecular-level 14C analysis of these compounds is particularly appealing because most industrial compounds are manufactured from petrochemicals (14C-free) and natural compounds should have “modern” or “contemporary” 14C levels. To investigate the source of DBP-Br4Cl2, we isolated 600 µg of this compound (150 µg of carbon) from marine animal extracts by employing gel permeation chromatography, Florisil column chromatography, and twodimensional preparative capillary gas chromatography. The purified DBP-Br4Cl2 was split into two samples (75 µg of carbon each) and analyzed by accelerator mass spectrometry for 14C content. The ∆14C values were -449‰ and -467‰, corresponding to conventional 14C ages of 4740 and 5000 years before present (BP), respectively. The presence of detectable 14C in the DBP-Br4Cl2 strongly points to at least a natural or biogenic source. However, these ∆14C values for DBP-Br4Cl2 are more depleted than expected for a recently synthesized natural product. Several explanations are discussed, but additional samples * Corresponding author phone: (508) 289-2316; fax: (508) 4572164; e-mail:
[email protected]. † Woods Hole Oceanographic Institution. ‡ University of California at San Diego. § National Wildlife Research Centre. | Institute of Ocean Sciences, Fisheries and Oceans Canada. ⊥ Carleton University. ∇ Present address: Food Research Division, Health Canada, Ottawa, Ontario, Canada K1A 0L2. 1992
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 7, 2004
Recent studies have detected two types of bipyrrolic halogenated organic compounds (HOCs) accumulating in various levels of marine food webs (Figure 1A,B). Tittlemier et al. (1-4) have identified and measured four halogenated 1,1′dimethyl-2,2′-bipyrroles (HDBPs) (Figure 1A) in zooplankton, fish, mammals, and sea birds in the Pacific, Indian, Arctic, and Atlantic Oceans at concentrations in the same range as those of polychlorobiphenyls (PCBs). Vetter and his colleagues (5-11) have found a related compound, heptachloro1′-methyl-1,2′-bipyrrole (Q1) (Figure 1B), in a variety of environmental and biological samples including air, foodstuffs, and human milk. While a marine bacterium was shown to produce a structurally similar compound, 3,3′,4,4′,5,5′hexabromo-2,2′-bipyrrole (Figure 1C) (12), the sources of the HDBPs and Q1 have not been identified. Since there is no record of direct industrial synthesis of the HDBPs or Q1, it is not known whether these compounds are unrecognized metabolites or byproducts of industrial compounds derived from anthropogenic activity or exclusively of biogenic origin. Constraining the sources and cycling of these and other HOCs of unknown origin is important for several reasons. First, their occurrence in an assortment of biota, including humans, indicates a widespread distribution in the environment. In addition, if truly naturally produced, these compounds have likely been present in the environment for a much longer time period than industrially synthesized HOCs. Therefore, HDBPs or Q1 may be an excellent subject to study the long-term fate of HOCs with similar physical properties, such as highly chlorinated PCBs. Conversely, if some of these compounds actually derive from anthropogenic activity, careful consideration would need to be given as to their source, mode of production, fate, and impact, and whether their emissions can or need to be controlled. In this study, we isolated one of the HDBPs found in marine animals (DBP-Br4Cl2; Figure 1A) and determined its radiocarbon (14C) content. Radiocarbon is produced in the atmosphere by collisions between cosmic-ray neutrons and 14 N, after which it is quickly oxidized to 14CO2. In addition, this signal is further enhanced by 14C released into the atmosphere from aboveground nuclear bomb testing in the 1950s and 1960s. Plants take up the 14CO2 for photosynthesis, and consequently they (and other members of the food web) reflect “modern” or “contemporary” levels of 14C. Once assimilation of 14C ceases (i.e., death of the organism), levels of 14C decrease through radioactive decay with a half-life of 5730 years. For this reason, 14C is an excellent tracer for establishing the origin of HOCs since all recent natural products should be prelabeled with at least some 14C. In contrast, products derived from petrochemical feedstock should contain no measurable 14C as petroleum forms over millions of years. This situation contrasts sharply for stable carbon isotope ratios (δ13C) where overlaps between natural and industrial signals can occur (13-14). We have recently produced data to support the premise that naturally produced HOCs contain contemporary levels of 14C (additional data are also presented in this manuscript) while most HOCs of industrial origin are 14C-free (11). The only exception of the latter was toxaphene (a chlorinated pesticide) as it was manufactured during the 1950s to 1970s by the photo10.1021/es030568i CCC: $27.50
2004 American Chemical Society Published on Web 03/02/2004
FIGURE 1. Structures of halogenated bipyrroles: (A) 1,1′-dimethyl3,3′,4,4′-tetrabromo-5,5′-dichloro-2,2′-bipyrrole (DBP-Br4Cl2), which is the most abundant halogenated 1,1′-dimethyl-2,2′-bipyrrole (HDBP) found in marine biota throughout the oceans (1-4), (B) heptachloro1′-methyl-1,2′-bipyrrole (Q1), which has been detected in oceanic biota, air, and human milk (5-11), and (C) 3,3′,4,4′,5,5′-hexabromo2,2′-bipyrrole synthesized by a marine bacterium (12). chlorination of camphene, an isomerization product of R-pinene extracted from pine tree stumps. However, a thorough search of the literature indicated that toxaphene was the only HOC industrially synthesized from a nonpetrochemical source. The results of this single 14C analysis of DBP-Br4Cl2 will be presented and discussed in the context of natural versus industrial sources.
Methods Extraction and Isolation of DBP-Br4Cl2. To obtain sufficient quantities of DBP-Br4Cl2 for 14C analysis (at least 25 µg of carbon), lipid extracts from several marine mammals and birds collected in or near the northern Pacific Ocean over the past 10 years were isolated according to the method described in Tittlemier et al. (2) and pooled. Tissues included blubber samples from killer whale (Orcinus orca), Dall’s porpoise (Phocoenoides dalli), and harbor porpoise (Phoceona phocoena), homogenized egg from black-footed albatross (Diomeda nigripes), and liver from bald eagles (Haliaeetus leucocephalus). Briefly, the samples were extracted with a 1:1 (v/v) solution of dichloromethane (DCM)/hexane after being ground with anhydrous Na2SO4. Triglycerides and other biological lipids were removed using gel permeation chromatography, and the lipid-free extracts were then separated on a Florisil column to yield an organohalogen fraction containing the DBP-Br4Cl2 as well as PCBs, 2,2-bis(pchlorophenyl)-1,1-dichloroethene (DDE), and other chlorinated pesticides. This fraction was then repeatedly injected onto a two-dimensional preparative capillary gas chromatograph programmed to trap (in a glass U-tube) the DBP-Br4Cl2 eluting from the second dimension column (Figure 2; 15). This approach has been used previously to isolate polycyclic aromatic hydrocarbons (PAHs) for 14C analysis (16, 17) except a second gas chromatograph oven was employed to make more refined separations. The first and second dimension columns were a CP-Sil 5CB (36 m × 0.53 mm i.d. × 0.50 µm film thickness) and DB-17ms (60 m × 0.53 mm i.d. × 0.50 µm film thickness), respectively. After ∼100 injections, the DBP-Br4Cl2 was recovered from the U-tube with DCM, and ∼5% was saved to test for purity by injection on a gas chromatograph equipped with a flame ionization detector (FID). Additional Naturally Produced HOCs. To expand on our previous work in which we found that naturally produced 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,5-dibromoanisole (Figure 3) had a modern 14C signal (13), we analyzed the 14C
content of five different HOCs isolated from red algae and worms (see structures in Figure 3). Violacene (18, 19), johnstonol (20), and 6β-hydroxyaplysistatin (21) were previously isolated from Plocamium violaceum, Laurencia johnstonii, and Laurencia filiformis (all are marine red algae that were collected in the 1970s from the Northeast Pacific Ocean), respectively. 2,3,4-Tribromopyrrole (22) was isolated and purified from Saccoglossus kowalevski, which was harvested from the top 20 cm of salt marsh sediments in Mashpee, MA, in July 2002. 2,4-Dibromophenol (22) was isolated and purified from Saccoglossus bromophenolosus living in the top 20 cm of sediment from Lowes Cove, ME, in June 2002. 14 C Analysis. Purified individual compounds were dissolved in DCM and added to precombusted quartz tubes (9 mm × 20 cm) (11, 16). The solvent was evaporated under a stream of nitrogen, and then ∼100 mg of copper oxide and several pieces of silver were added to each tube. The tubes were evacuated on a vacuum line, sealed, and combusted at 850 °C for 5 h, yielding carbon dioxide, water, and other combustion gases. The tubes were reattached to the vacuum line, and the carbon dioxide was isolated and purified through a series of cold traps and quantified by manometry. About 10% of the carbon dioxide was reserved for δ13C analysis by isotope ratio mass spectrometry. The remaining 90% was reduced to graphite (23). Targets of the graphite were pressed and mounted on target wheels for 14C analysis by accelerator mass spectrometry (AMS) at the National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) facility at Woods Hole Oceanographic Institution (WHOI), Woods Hole, MA (23). In this study, all 14C measurements are normalized to δ13C values of -25‰ and expressed as ∆14C values. The latter term is the per mille (‰) deviation from the international standard for 14C dating, Standard Reference Material 4990B “Oxalic Acid” (24). In this context, fossil carbon (14C-free) has a ∆14C of -1000‰ while recently photosynthesized materials are more enriched (∼0-200‰) depending on when and where the organism grew.
Results and Discussion To accurately measure the 14C content of the DBP-Br4Cl2, it was necessary to isolate a significant amount of the compound at a very high purity. Shown in Figure 4A is the GCFID chromatogram of the organohalogen extract from the combined animal tissue following Florisil column chromatography. The DBP-Br4Cl2 was the third most abundant HOC after DDE and trans-nonachlor. The other compounds that are not identified are mostly individual PCB congeners. After ∼100 injections of the latter extract into the two-dimensional preparative capillary gas chromatograph, ∼600 µg of the DBPBr4Cl2 (equivalent to 150 µg of carbon) was isolated at 98% purity (Figure 4B). The purified DBP-Br4Cl2 was split into two samples (75 µg of carbon each) and analyzed by AMS. The ∆14C values were -449‰ and -467‰, corresponding to conventional 14C ages of 4740 and 5000 years BP, respectively. The agreement between the two values was within typical AMS precision ((20‰) for small samples (
