Article pubs.acs.org/est
δ15N Enrichment Suggests Possible Source for Halogenated 1′Methyl-1,2′-bipyrroles (MBPs) Kristin C. Pangallo,†,‡,* Christopher M. Reddy,‡ Matthew Poyton,‡ Jakov Bolotin,§ and Thomas B. Hofstetter§ †
Department of Chemistry, Colgate University, Hamilton, New York 13346, United States Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, United States § Department of Environmental Chemistry, Eawag, Ueberlandstrasse 133, P.O. Box 611, 8600 Duebendorf, Switzerland ‡
ABSTRACT: Polyhalogenated 1′-methyl-1,2′-bipyrroles are natural products that biomagnify into upper trophic levels of marine food webs. Here we demonstrate that they are unusually enriched in 15N (δ15N from +19.3‰ to +28.1‰) relative to other biosynthetic organic compounds measured to date and the mammals from which the compounds were isolated. We argue the 15N enrichment likely stems from enriched precursors and/or fractionation during biosynthesis and is not from MBP degradation. We also consider possible sources of MBPs in light of these results.
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INTRODUCTION Halogenated 1′-methyl-1,2′-bipyrroles (MBPs) are a class of marine natural products that are composed of two pyrrole rings linked by an N,C1-bond and up to seven aromatic halogens (bromine or chlorine, Figure 1). Originally identified in marine
One method to infer the origins of a compound is by measuring its isotopic composition, which is imprinted during biosynthesis.10 For compounds that biomagnify such as MBPs, there is theoretically very little opportunity for isotope fractionation to occur since they should not undergo substantial metabolism and degradation in the environment.11 Hence, the isotopic signal of production, which relates to both the isotopic composition of precursor materials and the isotope fractionation of the biosynthetic reaction, should remain unchanged within the tissues of the higher trophic-level organisms.12 By isolating compounds from these higher trophic levels, we can determine the isotopic signature resulting from biosynthesis and thus gain insights into the origin of these persistent polyhalogenated compounds.13 It is well established the isotopic composition of a molecule is an average of the individual atoms within the structure, and that each of these positions can vary in their isotopic composition.10 Both MBP nitrogen atoms are pyrrolic (Figure 1) and pyrroles derive their nitrogen from amino acids,14 which obtain their nitrogen from isotopically homogeneous glutamate.15 This is unlike carbon atoms, for which there are multiple isotopically distinct pathways of fixation and incorporation into biomolecules.10 Thus, compound-specific N isotope analysis avoids the averaging inherent to similar analyses based on carbon and/or hydrogen. Here we employ compound-specific N isotope analysis to examine the origin of MBPs isolated from the blubber of three
Figure 1. The general structure of MBPs, where X represents Br, Cl, or H.
mammal blubber,1−3 MBPs biomagnify through marine food webs to reach the concentrations observed in blubber samples.4 For compounds to biomagnify they must be resistant to degradation and have octanol−water partition coefficients (Kow) greater than 105.5 These traits are shared by the persistent organic pollutants (POPs), which are also halogenated and have demonstrated negative impacts on environmental and human health.6 Due to these similarities, MBPs can be used as natural analogues to investigate the fate of POPs. The differences in origin between MBPs and POPs may limit this use unless MBP origins can be determined and accounted for in analyses comparing these compound classes. Although recent research has highlighted their geographic and trophic distributions,3,4,7−9 the origins and physiological role of MBPs remain elusive. © 2012 American Chemical Society
Received: Revised: Accepted: Published: 2064
September 9, 2011 January 18, 2012 January 23, 2012 January 23, 2012 dx.doi.org/10.1021/es203143c | Environ. Sci. Technol. 2012, 46, 2064−2070
Environmental Science & Technology
Article
common dolphins (Delphinus delphis), animals that have some of the highest MBP concentrations detected in environmental samples (1−2 μg g−1 lipid).4,9 We believe these are the first compound-specific N isotope analyses of biomagnified compounds.
We chose to isolate and enrich individual MBPs from the concentrated extract by preparative capillary gas chromatography (PCGC) prior to N isotope analysis by gas chromatography isotope-ratio mass spectrometry with a combustion interface (GC/IRMS). This choice was determined by the low N content of the MBPs (4.0−4.5 wt %%) and thus the large amounts of MBPs required to obtain adequate precision on the IRMS (see below, Quality Control). Moreover, by isolating the individual compounds prior to GC/IRMS, the quality of the chromatography did not limit our ability to make accurate measurements. This step was critical due to the large number of small, halogenated organic molecules contained in these extracts. The characteristics that make MBPs excellent natural analogues to the POPs (i.e., the similarities in their chemical and physical properties) also mean that their elution times are very similar. Thus, poor chromatography during GC-IRMS would be unacceptable with the pre-PCGC extract. The quantity of compound isolated was estimated by comparison to a synthetic standard of 1,3′MBP-Br7 using gas chromatography coupled to a flame ionization detector (GC/FID). The identities of the isolated compounds were confirmed with gas chromatography/electron capture negative ion mass spectrometry (GC/ECNI-MS). The details of the GC/ECNI-MS method and compound identification have been recently published.8 Nitrogen Isotope Analyses. Values for the δ15Nblubber of the dolphins were previously reported.4 Compound-specific nitrogen isotope analysis of MBPs was performed following a modified procedure of N isotope analysis in organic contaminants using GC/IRMS.17,18 The instruments used in our analyses were a Trace GC (ThermoFisher) coupled to an isotope ratio mass spectrometer (Delta V) via a combustion interface. Quality Control N Isotope Analysis. The low nitrogen content, high molecular weight (580−700 amu), and the large number of halogens per unit formula of MBP restricted accurate and precise δ15N measurements to small signal amplitudes between 100 and 500 mV (the background amplitude for m/z 28 was 25−28 mV) and 20−30 injections per oxidation/reduction reactor set in the combustion interface. Injections were thus limited to 3−6 nmoles of each MBP. As shown in Figure 2, δ15N values of a synthetic standard for halogenated bipyrroles determined by GC/IRMS (−4.7‰) were slightly more negative than reference measurements by elemental analyzer coupled to IRMS (−3.9 ± 0.2‰). The δ15N-values from more concentrated samples (GC/IRMS-peak amplitudes above 500 mV) suffered from unacceptably poor precision presumably due to incomplete conversion in the oxidation/reduction reactors of the combustion interface. Given these limitations and the observed typical increase of instrumental uncertainty with decreasing IRMS signal size,19 we assumed uncertainties of ±3‰ in δ15N for all measurements exhibiting peak amplitudes between 100 and 500 mV (Figure 2). This precision is a conservative estimate below current standards for GC/IRMS20 but sufficient for the detection of the substantial N isotope shifts in natural MBPs. Our uncertainty (±3‰) is also greater than the standard deviation of the triplicate analyses. To ensure accurate measurements of natural samples, δ15N-values of the synthetic standard DMBP-Cl6 were monitored after every 3 to 6 MBP measurements of blubber extracts and corrected for instrumental drift21 within ±3.7‰. Quality ControlExtraction and Purification Procedure. The entire extraction method was assessed to determine
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MATERIALS AND METHODS Samples. Blubber samples from three Delphinus delphis (Common dolphins) were acquired through the Cape Cod Stranding Network (CCSN) from dolphins stranded on Cape Cod, Massachusetts in 2004−2007 (CCSN numbers: CCSN04-218Dd, CCSN06-013Dd, and CCSN07-074Dd). CCSN04-218Dd was a maternally dependent male calf that was euthanized due to the stress of stranding. CCSN06-013Dd was a male in robust health that was euthanized after stranding due to deteriorating condition. CCSN07-074Dd was an adult female in robust health that was lactating at the time of stranding but died at the stranding site. Chemicals. All solvents were GC Resolve grade and purchased from Fisher Scientific. Our standards, 2,2′,3,4,4′,5,5′-heptabromo-1′-methyl-1,3′-bipyrrole (1,3′-MBPBr7) and 3,3′,4,4′,5,5′-hexachloro-1,1′-dimethyl-2,2′-bipyrrole (DMBP-Cl6), were synthesized for us by the laboratory of Gordon Gribble (Dartmouth College). Details on our nomenclature system are available elsewhere8,9 and consistency between these publications allows results to be easily compared. Briefly, because halogen position has yet to be determined for most MBPs, individual congeners are designated by their halogen content plus a lower case letter. The letter refers to the elution order resulting from gas chromatography performed with a DB-XLB column. The MBPs discussed here are the most abundant hexabromochloro-MBP congener (MBP-Br6Cl-b), and the most abundant hexabromoMBP congener (MBP-Br6-b). As the structure of the perbrominated congener is known, we use the acronym developed by Vetter et al. for 2,3,3′,4,4′,5,5′-heptabromo-1′methyl-1,2′-bipyrrole (Br7-MBP-79).16 MBP Isolation from Blubber. The blubber was received, stored as frozen slabs, and thawed prior to processing. It was then homogenized with hexane and filtered to a clear, yellow liquid, referred to as the total lipid extract (TLE). The solvent was removed, and the oil was stored at −20 °C. To isolate large enough quantities of the molecules of interest for compound-specific nitrogen isotope analysis, 250 g of oil was used for each sample. We employed gel permeation chromatography (GPC) to isolate the target compounds from the lipids in our samples, as highly brominated MBPs are not stable during acid degradation of lipids.3 A 10 g aliquot of each sample was applied to the top of a 3 cm (o.d.) column, which was packed with 100 g of SX-8 BioBeads (∼45 cm, uncompressed). We used a mobile phase of 1:1 dichloromethane:hexane and collected two fractions. The first fraction (0−150 mL) contained ∼70% of the lipids and was discarded; the second fraction (150−400 mL), containing the remaining lipids and target compounds, was reduced in volume by rotary evaporation. This procedure was repeated with the remaining oil from the initial 250 g sample and then the second fractions were combined and reapplied to the GPC column in 10 g aliquots. This was performed until
