Challenges in the Analysis of Novel Flame Retardants in Indoor Dust: Results of the INTERFLAB 2 Interlaboratory Evaluation.

The Interlaboratory Study of Novel Flame Retardants (INTERFLAB 2) was conducted by 20 laboratories in 12 countries to test the precision and accuracy of the analysis of 24 "novel" flame retardants (NFRs). Laboratories analyzed NFRs in injection-ready test mixtures, in extracts of residential dust, and in residential dust to evaluate the influence of dust handling and extraction. For test mixtures, mean reported concentrations of PBT, PBEB, EH-TBB, TBBPA, TBDP-TAZTO, TBOEP, α-TBCO, ß-DBE-DBCH, and total HBCDD differed by >25% relative to reference values. Coefficients of variation were higher in dusts/dust extracts than in test mixtures. Concentrations among laboratories ranged over 3-4 orders of magnitude for HBB, TBP-DBPE, TBP-AE, and TDCIPP in dust extracts and dusts. Most laboratories produced repeatable dust concentrations, but differences reported in the literature among laboratories of <70% could be due to analytical variability, and the attribution of such differences to other causes should be made with caution. Most variations in accuracy and precision were introduced by matrix effects and/or sample processing, rather than instrumental analysis. We recommend recovery correction to improve accuracy. There is a need to improve analytical methods and to validate methods on complex matrices such as standard reference materials for dust or spiked matrices.

Characterization of Nine Isomers in Commercial Samples of Perfluoroethylcyclohexanesulfonate and of Some Minor Components Including PFOS Isomers.

Electrochemical fluorination of 4-ethylbenzenesulfonyl halides produces a mixture of compounds that has found extensive use as an erosion inhibitor in aircraft hydraulic fluids. This paper reports a study of the composition of commercial samples of this material from two industrial scale manufacturers in terms of the structures and relative concentrations of their components, the major of which is perfluoroethylcyclohexanesulfonate (PFECHS). Fractionation of one of these mixtures by column chromatography produced fractions in which all significant components were of sufficient purity to allow assignment of their structures by 19F NMR spectroscopy. Assessment of the relevant signals in the 19F NMR spectra of the commercial mixtures by integration revealed the presence of 14 constituents at levels ≥0.5% in terms of molar contributions. Ten of these involve five pairs of geometric isomers, including cis- and trans-PFECHS which accounted for between 55% and 60% of the components present. Three constituents were determined to be isomers of perfluorooctanesulfonate (PFOS), two branched and the other linear PFOS itself. The availability of samples of the various components also allowed us to identify the compounds responsible for the peaks observed when the commercial samples were analyzed by LC/MS using either C18 or perfluorophenyl stationary phases.

Dechlorinated Analogues of Dechlorane Plus.

Degradation products of the chlorinated additive flame retardant Dechlorane Plus (DP) have been discovered globally. However, the identity of many of these species remains unknown due to a lack of available analytical standards, hindering the ability to quantitatively measure the amounts of these compounds in the environment. In the present study, synthetic routes to possible dechlorinated DP derivatives were investigated in an effort to identify the environmentally significant degradation products. The methano-bridge chlorines of anti- and syn-DP were selectively replaced by hydrogen atoms to give six new hydrodechlorinated DP analogues. The identity and absolute configuration of all of these compounds were confirmed by GC-MS, NMR spectroscopy, and X-ray diffraction studies. These compounds were observed in sediment samples from streams and rivers in relatively rural areas of Ontario and are thus environmentally relevant.

Enantioselective Analytical- and Preparative-Scale Separation of Hexabromocyclododecane Stereoisomers Using Packed Column Supercritical Fluid Chromatography.

Hexabromocyclododecane (HBCDD) is an additive brominated flame retardant which has been listed in Annex A of the Stockholm Convention for elimination of production and use. It has been reported to persist in the environment and has the potential for enantiomer-specific degradation, accumulation, or both, making enantioselective analyses increasingly important. The six main stereoisomers of technical HBCDD (i.e., the (+) and (-) enantiomers of α-, ß-, and γ-HBCDD) were separated and isolated for the first time using enantioselective packed column supercritical fluid chromatography (pSFC) separation methods on a preparative scale. Characterization was completed using published chiral liquid chromatography (LC) methods and elution profiles, as well as X-ray crystallography, and the isolated fractions were definitively identified. Additionally, the resolution of the enantiomers, along with two minor components of the technical product (δ- and ε-HBCDD), was investigated on an analytical scale using both LC and pSFC separation techniques, and changes in elution order were highlighted. Baseline separation of all HBCDD enantiomers was achieved by pSFC on an analytical scale using a cellulose-based column. The described method emphasizes the potential associated with pSFC as a green method of isolating and analyzing environmental contaminants of concern.

Characterization and Biological Potency of Mono- to Tetra-Halogenated Carbazoles.

This paper deals with the characterization and aryl hydrocarbon receptor (AhR) agonist activities of a series of chlorinated, brominated, and mixed bromo/chlorocarbazoles, some of which have been identified in various environmental samples. Attention is directed here to the possibility that halogenated carbazoles may currently be emitted into the environment as a result of the production of carbazole-containing polymers present in a wide variety of electronic devices. We have found that any carbazole that is not substituted in the 1,3,6,8 positions may be lost during cleanup of environmental extracts if a multilayer column is utilized, as is common practice for polychlorinated dibenzo-p-dioxin (dioxin) and related compounds. In the present study, (1)H NMR spectral shift data for 11 relevant halogenated carbazoles are reported, along with their gas chromatographic separation and analysis by mass spectrometry. These characterization data allow for confident structural assignments and the derivation of possible correlations between structure and toxicity based on the halogenation patterns of the isomers investigated. Some halogenated carbazoles exhibit characteristics of persistent organic pollutants and their potential dioxin-like activity was further investigated. The structure-dependent induction of CYP1A1 and CYP1B1 gene expression in Ah-responsive MDA-MB-468 breast cancer cells by these carbazoles was similar to that observed for other dioxin-like compounds, and the magnitude of the fold induction responses for the most active halogenated carbazoles was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 2,3,6,7-Tetrachlorocarbazole was one of the most active halogenated carbazoles and, like TCDD, contains 4 lateral substituents; however, the estimated relative effect potency for this compound (compared to TCDD) was 0.0001 and 0.0032, based on induction of CYP1A1 and CYP1B1 mRNA, respectively.

Dechloranes 602, 603, 604, Dechlorane Plus, and Chlordene Plus, a newly detected analogue, in tributary sediments of the Laurentian Great Lakes.

A chlorinated compound (Chlordene Plus, CP), structurally related to Dechloranes (Dec) 602, 603, 604, and Dechlorane Plus (DP), was identified, and concentrations and spatial trends of Dec 602, 603, 604, CP, and DP in tributary sediments of the Laurentian Great Lakes are reported. The dechloranes were widely detected with their concentrations varying considerably across the Great Lakes basin. Spatial trends of Dec 602, 604, and DP in Canadian tributary sediments were similar to that of BDE 209, which suggested these flame retardant chemicals in tributaries were associated with industrial and urban areas. The highest concentrations of Dec 602, 604, and DP observed in tributaries of the Niagara River confirmed that past or ongoing manufacturing of these compounds at plants along the river were important sources to Lake Ontario. Dec 603 was detected in technical products of aldrin and dieldrin, and its spatial trend was consistent with historic pesticide usage. Similarly, CP was detected in technical products of chlordene and chlordane, and it was found in higher concentrations in sediments near urban areas, possibly related to past chlordane use in home termite control.

Species-specific regulation of PXR/CAR/ER-target genes in the mouse and rat liver elicited by o, p'-DDT.

BACKGROUND: Dichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data. RESULTS: Rats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT. CONCLUSION: Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

o,p'-DDT elicits PXR/CAR-, not ER-, mediated responses in the immature ovariectomized rat liver.

Technical-grade dichlorodiphenyltrichloroethane (DDT) is an agricultural pesticide and malarial vector control agent that has been designated a potential human hepatocarcinogen. The o,p'-enantiomer exhibits estrogenic activity that has been associated with the carcinogenicity of DDT. The temporal and dose-dependent hepatic estrogenicity of o,p'-DDT was investigated using complementary DNA microarrays in immature ovariectomized Sprague-Dawley rats with complementary histopathology and tissue-level analysis. Animals were gavaged with 300 mg/kg o,p'-DDT either once or once daily for 3 consecutive days. Liver samples were examined 2, 4, 8, 12, 18, or 24 h after a single dose or following three daily doses. For dose-response studies, a single dose of 3, 10, 30, 100, or 300 mg/kg body weight o,p'-DTT was administered for 3 consecutive days. Genes associated with drug metabolism (Cyp2b2 and Cyp3a2), the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), cell proliferation (Ccnd1, Ccnb1, Ccnb2, and Stmn1), and oxidative stress (Gclm and Hmox1) were significantly induced. Cyp2b2 exhibited dose-dependent regulation and was significantly induced across all time points, while cell proliferation- and oxidative stress-related genes exhibited transient induction. The induction of Cyp2b2 and Cyp3a2 mRNA levels suggest PXR/CAR activation, consistent with expression of genes associated with oxidative stress. Few genes known to be estrogen receptor (ER) regulated were differentially expressed when compared to the hepatic gene expression profile elicited by ethynyl estradiol in immature ovariectomized C57BL/6 mice using the same study design and analysis methods. These data indicate that o,p'-DDT elicits PXR/CAR-, not ER-, mediated gene expression in the rat liver. Based on the species-specific differences in CAR regulation, the extrapolation of rodent DDT hepatocarcinogenicity to humans warrants further investigation.

Comparative toxicogenomic examination of the hepatic effects of PCB126 and TCDD in immature, ovariectomized C57BL/6 mice.

Polychlorinated biphenyls are persistent environmental pollutants that elicit a wide range of effects in humans and wildlife, mediated by the aryl hydrocarbon receptor. 3,3',4,4',5-pentachlorobiphenyl (PCB126) is the most potent congener with relative effect potencies ranging from 0.0026 to 0.857, and a toxic equivalency factor (TEF) of 0.1 set by an expert panel of the World Health Organization. In this study, the hepatic effects elicited by 300 microg/kg PCB126 were compared with 30 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature, ovariectomized female C57BL/6 mice. Comprehensive hepatic gene expression analyses with complementary histopathology, high-resolution gas chromatograph/high-resolution mass spectrometer tissue analysis, and clinical chemistry were examined. For temporal analysis, mice were orally gavaged with PCB126 or sesame oil vehicle and sacrificed after 2, 4, 8, 12, 18, 24, 72, 120, or 168 h. In the dose-response study, mice were gavaged with 0.3, 1, 3, 10, 30, 100, 300, 1000 microg/kg PCB126, 30 or 100 microg/kg TCDD and sacrificed after 72 h. 251 and 367 genes were differentially expressed by PCB126 at one or more time points or doses, respectively, significantly less than elicited by TCDD. In addition, there was less vacuolization and necrosis, and no immune cell infiltration, despite comparable or higher TEF-adjusted hepatic PCB126 levels. The functional annotation of differentially expressed genes was consistent with the observed histopathology. Collectively, the data indicate that 300 microg/kg PCB126 elicited a subset of weaker effects compared with 30 microg/kg TCDD in immature, ovariectomized C57BL/6 mice.

Separation and fluorine nuclear magnetic resonance spectroscopic (19F NMR) analysis of individual branched isomers present in technical perfluorooctanesulfonic acid (PFOS).

The production of perfluoroalkylsulfonate (PFOS) derivatives from linear alkyl precursors using electrochemical fluorination is not a clean process but, instead, gives complex mixtures. This study reports the isolation and (19)F NMR characterization of eleven perfluorooctanesulfonate isomers from a commercial mixture. This allowed the quantification of the individual CF(3) branched isomers that predominate in technical PFOS.

Some issues relating to the use of perfluorooctanesulfonate (PFOS) samples as reference standards.

Samples of potassium perfluorooctanesulfonate (PFOSK) from three suppliers were analyzed by LC-ESI-MS/MS for purity and by LC-ESI-MS for the percentage of linear isomer present. Our data indicated that the purity ranged from 80% to 98% and the percentages of linear isomer from 67% to 79%. The proportion of branched isomers present in the samples was also estimated using (19)F NMR. These results agreed quite closely with those found by LC-ESI-MS indicating that there is essentially no difference in overall SIM response factor for the branched isomers vs. that of the linear isomer. Several further observations relevant to the use of standards when analyzing for PFOS were encountered during this study. It appears unlikely that matrix effects attributable to the cation (sodium or potassium) present in PFOSNa or PFOSK internal standards is an issue. In seeking potential matrix effects, it was found that the chromatography was improved substantially when the standard was injected as a solution in 80:20 methanol/water rather than 100% methanol. Notably, in concert with the improvement in chromatography, an increase of about 10% in response was observed. In some closely related studies, when (18)O(2) mass-labeled perfluorohexanesulfonate was used as an internal standard, the actual and theoretical concentration ratios matched closely those for related native sulfonates as long as they did not co-elute. However, when they did co-elute, the peak intensities of the native species were enhanced by about 5%, while those of the labeled compound were suppressed by a similar amount. If this effect were not taken into account, the concentration of the native would be inflated by 10%.

Identification of the minor components of Great Lakes DE-71 technical mix by means of 1H NMR and GC/MS.

The production of technical penta-BDE products such as Great Lakes DE-71 is not a clean process but, instead, gives complex mixtures of various BDE congeners. This study reports the verification of the structures of many of the BDE congeners in Great Lakes DE-71 using (1)H NMR and/or GC/MS. In total, 24 BDE congeners, including nine (tetra-BDEs 42, 48, 51, and 91; penta-BDEs 102, 104, and 119; hexa-BDEs 149 and 155) which had not been reported previously, were identified in this technical mix by (1)H NMR. The quantification of these congeners was realized by two independent methods: (1)H NMR spectroscopy in combination with HRGC/LRMS and isotopic dilution and HRGC/HRMS analysis. The values obtained compare well between methods, and with data produced in earlier studies.

Electrolytic debromination of PBDEs in DE-83 technical decabromodiphenyl ether.

Electrochemical debromination of the commercial decabromodiphenyl ether flame retardant DE-83 in partly aqueous tetrahydrofuran (THF) solution gave lower brominated congeners by sequential loss of bromine atoms. Hydrodebromination was most facile for the most heavily brominated congeners. It involves initial electron transfer and proton transfer from water, rather than hydrogen atom abstraction from THF, as shown by experiments with deuterated water. The product distribution from electrolysis involves preferential loss of bromine meta- and para- to the ether linkage, comparable with the products of metabolism of BDE-209 in various organisms. Significantly, the environmentally relevant congeners BDE-47, BDE-99, and BDE-154 were not major products of debromination of BDE-209 by the electron transfer mechanism.

Nuclear magnetic resonance spectral characterization and semi-empirical calculations of conformations of alpha- and gamma-1,2,5,6,9,10-hexabromocyclododecane.

The full 1H and 13C NMR spectral characterization of alpha- and gamma-1,2,5,6,9,10-hexabromocyclododecane (HBCD) is reported in this paper. The use of various NMR experiments, an analysis of the magnitude of the NMR chemical shifts and coupling constants, and computer modeling has enabled the visualization of the three-dimensional structures for both the alpha- and gamma-diastereomers. This information may provide insight into the different behavior of the alpha- and gamma-HBCD diastereomers in the environment.

Coupling of supercritical fluid chromatography to mass spectrometry for the analysis of Dechlorane Plus: Examination of relevant negative ion atmospheric pressure chemical ionization mechanisms.

During an investigation of the potential associated with coupling packed column supercritical fluid chromatography (pSFC) to mass spectrometry for the analysis of Dechlorane Plus and related compounds, it was found that negative ion atmospheric pressure chemical ionization (APCI) was a promising ionization technique. In the course of maximizing the responses associated with the target analytes, it proved useful to examine some aspects of the complex nature and reactivity of the corona discharge plasma generated to explain the observed ionization products. Various dopants/reagents were screened for both APCI and atmospheric pressure photoionization (APPI) in negative ion mode and mechanisms of ionization involving superoxide were elucidated based on the results obtained. Superoxide formation was found to be temperature dependent and directly related to the intensity of the ion cluster [M-Cl+O]- obtained for the target DP analytes. Furthermore, triethylamine was identified as a reagent capable of suppressing unwanted side reactions during the ionization process and maximizing response associated with the analytes of interest. The applicability of pSFC-APCI/MS for the separation and detection of Dechlorane Plus and related compounds was demonstrated by analyzing Lake Ontario sediment and comparing the results with values reported in the scientific literature.

Examination of technical mixtures of halogen-free phosphorus based flame retardants using multiple analytical techniques.

The application of phosphorus based flame retardants as replacements for commonly used halogenated flame retardants has been gaining interest due to the possibility that these compounds may have a less significant impact on human and environmental health. Unfortunately, little is known about the chemical compositions of many of the technical products (which often are mixtures) and a single separation technique for concurrent analysis of these types of compounds has not been identified. This paper reports the results of an investigation into the constituents of three halogen free organophosphate flame retardants (OPFRs), resorcinol bis(diphenyl phosphate) (RDBPP), bisphenol A bis(diphenyl phosphate) (BPA-BDPP), and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO). The major components of commercial samples of RDBPP and BPA-BDPP were isolated by preparative TLC and characterized by NMR. A commercial sample of DOPO was found to be essentially pure, but its analysis is complicated by the fact that it can exist in ring-open and ring-closed forms. With the structures of the components confirmed by NMR, multiple analytical separation techniques (gas chromatography (GC), liquid chromatography (LC), and packed column supercritical fluid chromatography (pSFC)) were investigated for the analysis of these three technical products. Packed column supercritical fluid chromatography allows the separation of the components of all three OPFRs, including the two forms of DOPO, in a single run.

Comparative toxicogenomic analysis of the hepatotoxic effects of TCDD in Sprague Dawley rats and C57BL/6 mice.

In an effort to further characterize conserved and species-specific mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated toxicity, comparative temporal and dose-response microarray analyses were performed on hepatic tissue from immature, ovariectomized Sprague Dawley rats and C57BL/6 mice. For temporal studies, rats and mice were gavaged with 10 or 30 microg/kg of TCDD, respectively, and sacrificed after 2, 4, 8, 12, 18, 24, 72, or 168 h while dose-response studies were performed at 24 h. Hepatic gene expression profiles were monitored using custom cDNA microarrays containing 8567 (rat) or 13,361 (mouse) cDNA clones. Affymetrix data from male rats treated with 40 microg/kg TCDD were also included to expand the species comparison. In total, 3087 orthologous genes were represented in the cross-species comparison. Comparative analysis identified 33 orthologous genes that were commonly regulated by TCDD as well as 185 rat-specific and 225 mouse-specific responses. Functional annotation using Gene Ontology identified conserved gene responses associated with xenobiotic/chemical stress and amino acid and lipid metabolism. Rat-specific gene expression responses were associated with cellular growth and lipid metabolism while mouse-specific responses were associated with lipid uptake/metabolism and immune responses. The common and species-specific gene expression responses were also consistent with complementary histopathology, clinical chemistry, hepatic lipid analyses, and reports in the literature. These data expand our understanding of TCDD-mediated gene expression responses and indicate that species-specific toxicity may be mediated by differences in gene expression which may help explain the wide range of species sensitivities and will have important implications in risk assessment strategies.

Structure-dependent Ah receptor agonist activities of chlorinated biphenylenes.

Polychlorinated biphenylenes (PCBP) have been identified as combustion by-products that bind the aryl hydrocarbon receptor (AhR) and exhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like activity. This study investigates the Ah-responsiveness of 2,3,6,7-tetrachlorobiphenylene (2,3,6,7-CBP), 2,3,6-CBP, 2,3-CBP and 2-CBP in breast cancer cells. MCF-7 or ZR-75 cells were treated with different concentrations (1-100 nM) of the compounds alone to determine their activity as inducers of CYP1A1 protein expression or luciferase activity in cells transfected with a construct (pDRE(3)) containing three tandem dioxin responsive elements (DREs) linked to a luciferase reporter gene. In both assays, the order of potency was 2,3,6,7-CBP>2,3,6-CBP>2,3-CBP approximately 2-CBP, and 2,3,6,7-CBP and TCDD were equipotent. Similar results were also observed in an antiestrogenic assay in MCF-7 cells, confirming the high AhR agonist activity of 2,3,6,7-CBP in breast cancer cells.

Temporal and dose-dependent hepatic gene expression patterns in mice provide new insights into TCDD-Mediated hepatotoxicity.

In an effort to further characterize the mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated toxicity, comprehensive temporal and dose-response microarray analyses were performed on hepatic tissue from immature ovariectomized C57BL/6 mice treated with TCDD. For temporal analysis, mice were gavaged with 30 microg/kg of TCDD or vehicle and sacrificed after 2, 4, 8, 12, 18, 24, 72, or 168 h. Dose-response mice were gavaged with 0, 0.001, 0.01, 0.1, 1, 10, 100, or 300 microg/kg of TCDD and sacrificed after 24 h. Hepatic gene expression profiles were monitored using custom cDNA microarrays containing 13,362 cDNA clones. Gene expression analysis identified 443 and 315 features which exhibited a significant change at one or more doses or time points, respectively, as determined using an empirical Bayes approach. Functional gene annotation extracted from public databases associated gene expression changes with physiological processes such as oxidative stress and metabolism, differentiation, apoptosis, gluconeogenesis, and fatty acid uptake and metabolism. Complementary histopathology (H&E and Oil Red O stains), clinical chemistry (i.e., alanine aminotransferase [ALT], triglyceride [TG], free fatty acids [FFA], cholesterol) and high-resolution gas chromatography/mass spectrometry assessment of hepatic TCDD levels were also performed in order to phenotypically anchor changes in gene expression to physiological end points. Collectively, the data support a proposed mechanism for TCDD-mediated hepatotoxicity, including fatty liver, which involves mobilization of peripheral fat and inappropriate increases in hepatic uptake of fatty acids.

Halogenated indigo dyes: a likely source of 1,3,6,8-tetrabromocarbazole and some other halogenated carbazoles in the environment.

In recent years, a number of halogenated carbazoles have been detected in environmental samples. These emerging contaminants have been shown to be persistent and possess dioxin-like toxicological potential. The goal of this research was to examine the literature to determine likely anthropogenic origin(s) of halogenated carbazoles in the environment. The scientific literature indicated a number of pathways by which 1,3,6,8-tetrabromocarbazole could form in the manufacture of 5,5',7,7'-tetrabromoindigo. The U.S. production history of 5,5',7,7'-tetrabromoindigo correlates well with the concentration rise, decline, and disappearance of 1,3,6,8-tetrabromocarbazole in dated Lake Michigan sediments. Additionally, other halogenated carbazoles that have been found in environmental sediments can be explained by the production of other halogenated indigo dyes. 1,8-dibromo-3,6-dichlorocarbazole can be accounted for by the manufacture of 7,7'-dibromo-5,5'-dichloroindigo, while 1,3,6,8-tetrachlorocarbazole was found at relatively high concentration near the outfall of a U.S. manufacturer of 5,5',7,7'-tetrachloroindigo. Carbazoles containing an iodo-substituent can be explained by the use of iodine as a catalyst in the manufacture of halogenated indigo dyes. 3,6-Dichlorocarbazole measured in soils and dibromocarbazoles measured in more recently deposited sediments are not easily rationalized on the basis of an indigo related source and may be related to other anthropogenic sources or natural origins.