Flame Retardant Metabolites in Addled Bald Eagle Eggs from the

Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/estlcu

Flame Retardant Metabolites in Addled Bald Eagle Eggs from the Great Lakes Region William A. Stubbings,† Jiehong Guo,† Kendall Simon,‡ Kevin Romanak,† William Bowerman,‡ and Marta Venier*,† †

School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana 47405, United States Department of Environmental Science and Technology, University of Maryland, College Park, Maryland 20742, United States

‡

S Supporting Information *

ABSTRACT: Organophosphate esters (OPEs) and other alternative flame retardants including 2-ethylhexyl-2,3,4,5tetrabromobenzoate (EHTBB) and bis(2-ethylhexyl) tetrabromophthalate (BEHTBP) are ubiquitous in the Great Lakes region, having been detected in air, water, and biota samples. In a recent study, however, we showed that concentrations of six OPEs in eagle eggs were generally low (geometric mean 24 ng/g ww), and the two main Firemaster (FM) components, EHTBB and BEHTBP, were below limits of detection. We therefore hypothesized that the low levels of these compounds in bald eagles may be due to a potential rapid metabolic transformation. We measured metabolites of triaryl and trialkyl phosphates and brominated Firemaster (FM) flame retardants in 21 addled bald eagle (Haliaeetus leucocephalus) eggs from 2000 to 2012 from the Michigan Bald Eagle Biosentinel Program archive. Sampling sites were divided into two groups: inland (IN) and Great Lakes (GL) based on breeding areas. Results suggest that the most abundant metabolites in the eggs are 2,3,4,5tetrabromobenzoic acid (TBBA) (n.d. − 330 ng/g ww), bis(2-chloroethyl) phosphate (BCEP) (0.38−26 ng/g ww), and bis(2,3dibromopropyl) phosphate (BDBPP) (n.d. − 45 ng/g ww). Detection frequencies ranged from 67% for mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) to 100% for most of the other compounds. The relative abundances for these parent/ metabolite pairs indicate that the majority of the OPEs can be readily metabolized at various rates, depending on the specific compound.

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INTRODUCTION

in eggs, serum, maternal muscle, maternal fat, and feathers of birds from the Northern Hemisphere.9−16 Concentrations of six OPEs previously measured in the same addled bald eagle eggs were generally low (geometric mean 24 ng/g ww) and the two main FM components, 2-ethylhexyl2,3,4,5-tetrabromobenzoate (EHTBB) and bis(2-ethylhexyl) tetrabromophthalate (BEHTBP), were below limits of detection.17 This, together with similarly low concentrations reported for bird tissue samples from other studies,18 led to the hypothesis of potential rapid metabolic transformation of these compounds.19 Relatively little information exists regarding the biological effect and toxicity of OPE metabolites. In a 36 h exposure study of chicken embryonic hepatocyte cultures to 10 μM triphenyl phosphate (TPhP) or its primary metabolite, diphenyl phosphate (DPhP), DPhP altered more genes (9 of 27) compared to the parent compound (3 of 27); all significantly down-regulated (p < 0.05, fold change >1.5).20 In total, five

Bald eagles (Haliaeetus leucocephalus) are apex predators, occupying the highest trophic level, and as such are more susceptible to bioaccumulating high concentrations of persistent organic pollutants. Bald eagles are opportunistic predators, hunting for live prey consisting of water fowl and fish, in addition to scavenging on carrion. In 1999, under the Clean Michigan Initiative, the Michigan Department of Environmental Quality implemented the Michigan Bald Eagle Biosentinel Monitoring Program in which bald eagles are used to monitor trends of persistent chemicals. Recent studies have shown that OPEs and FM components are the most widely used flame retardants (FR) in polyurethane foams from baby products to residential furniture in the United States.1,2 Further, high concentrations of these compounds have been detected in the atmosphere,3,4 tributaries,5,6 and fish7 of the Great Lakes. The ubiquitous nature of these compounds across the Great Lakes infer that avian species breeding in the region are inevitably exposed. Flame retardants, polychlorinated biphenyls, and pesticides have previously been detected in bald eagle plasma collected from the Great Lakes region.8 Additionally, OPEs have been detected in low concentrations © XXXX American Chemical Society

Received: March 23, 2018 Revised: April 26, 2018 Accepted: May 2, 2018

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DOI: 10.1021/acs.estlett.8b00163 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

Letter

Environmental Science & Technology Letters

Table 1. List of Target OPE and FM Metabolite Structures, Abbreviations, Molecular Weights, CAS No., Molecular Formulas, and MRM Transitions

Biosentinel Monitoring Program archive. The addled eggs were collected from bald eagle nests over a 12 year period (2000− 2012, inclusive). The goals of this study were 2-fold: (1) to develop a working method for the analysis of OPE and FM metabolites in avian egg matrix without the need for derivatization and (2) to assess the levels of OPE and FM metabolites found in 21 addled bald eagle eggs collected from the Great Lakes region and compare them to the levels of their parent compounds.

gene pathways were altered, including genes associated with the thyroid hormone pathway (DIO1, THRSP), which is critically important to normal central nervous system development, growth, and metabolism in avian species.20 BEHTBP can be metabolized into mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) by carboxylesterase. In a study on toxicity mechanisms of human vascular endothelial cells, TBMEHP inhibited cell growth, as well as induced cell cycle arrest and apoptosis via altering cell cycle regulatory genes expression p53 while initiating mitochondria mediated apoptosis signal pathways.21 Moreover, TBMEHP was found to be more toxic than its parent compound.21 The potential toxicological effects of OPE and FM metabolites, as well as the ubiquity of their parent compounds in the environment, suggests that they could be potential threats to recovering bald eagle populations. In this study, we selected 21 addled egg samples from the Michigan Bald Eagle

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MATERIALS AND METHODS Chemicals. This study targeted seven OPEs metabolites [bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2-propyl) phosphate (BCIPP), bis(2,3-dibromopropyl) phosphate (BDBPP), bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), di-n-butyl phosphate (DnBP)] and two FM metabolites [mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) and B

DOI: 10.1021/acs.estlett.8b00163 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Letters

the analytical methods and standards can be found in the SI and in Table S2. A MS/MS method using multiple reaction monitoring (MRM) was used with the source operated in negative electrospray ionization (N-ESI) mode. Details of instrument parameters and monitored ions are reported in the SI. Quality Assurance/Quality Control. A matrix spike and lab blank were performed simultaneously with each set of egg samples. The matrix spike comprised a mixture of both the native compounds and IS at ca. 100 ng for both OPE and FM metabolites. Matrix spikes to both blank samples and homogenized chicken eggs were utilized in method development to assess the effect of matrix interferences (Table S3). Mean matrix spike recoveries were in the range 58%−108%, and mean IS recoveries were in the range 52%−104%. Lab blanks were used to evaluate contamination from laboratory procedures. The concentrations of the target compounds in each sample were compared to the mean concentrations in the blanks. Blank levels for all compounds were 90%), suggesting widespread FR contamination in these birds. There was a wide range of concentrations in both TBBA (nondetect − 330 ng/g ww) and TBMEHP (nondetect − 20 ng/g ww) in the eggs, as represented by the large boxes in Figure 1. The geometric mean concentration of TBBA was 24 ± 20 ng/g ww, and TBMEHP concentrations were generally 1 order of magnitude less (geometric mean 2.1 ± 1.9 ng/g ww). The detection frequency for TBMEHP was also lower than that D

DOI: 10.1021/acs.estlett.8b00163 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

Letter

Environmental Science & Technology Letters

compound. Some of the OPEs are not readily metabolized (i.e., TPhP and TnBP); hence, they have the ability to bioaccumulate within the food chain, with the potential to biomagnify. However, the metabolites of compounds that are readily metabolized may have a greater toxicity potential. Considering that very little information is available on the toxicity of these compounds, these findings are concerning and warrant further research.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.estlett.8b00163. Details on sampling and storage, chemicals used, analytical methods including instrumental parameters, and QA/QC. Individual concentrations for all target compounds in all samples and summary statistics. (PDF)

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Figure 2. Median OPE and FM parent and metabolite percent profiles for bald eagle (Haliaeetus leucocephalus) eggs. Note that the plot is not normalized for detection frequency. Detection frequencies for the parent compounds are TDBPP 14%, TPhP 95%, TnBP 100%, TCIPP 90%, TDCIPP 62%, TCEP 52%, TBOEP 0%, EHTBB 0%, and BEHTBP 0%. Detection frequencies for the metabolites are given in Table S4.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: 1-812-855-1005. ORCID

Marta Venier: 0000-0002-2089-8992 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for this study was provided by the Michigan Department of Environmental Quality under the Clean Michigan Initiative and the U.S. Fish and Wildlife Service. The authors gratefully acknowledge helpful and insightful discussions with Jonathan Karty (Indiana University, IN, USA), Mohamed A.-E. Abdallah (University of Birmingham, UK), and Syrago-Styliani E. Petropoulou (Department of Toxic Substances Control, CA, USA) on the development of the LC-MS/ MS method for OPE metabolites. Additionally, we thank Heather M. Stapleton (Duke University, NC, USA) for the generous donation of the 13C-DPhP standard synthesised at the Small Molecule Synthesis Facility at Duke University (Durham, NC). We also thank David Best with Fish and Wildlife Service, East Lansing, MI for his assistance in collection of the bald eagle eggs.

(n = 3). Additionally, the instrument detection limit for TDBPP (3.7 ng) via GC-MS analysis is relatively high. The low conversion rate or efficiency of these three parent compounds contradicts the common assumption that OPE flame retardants are not bioaccumulative. The second group of compounds includes the pairs BCIPP/ TCIPP, BDCIPP/TDCIPP, and BCEP/TCEP, which are at the center of the plot. For these pairs, the abundances of the parent and metabolites are relatively similar, with the parent compound representing 30%−50% of the total. For these pairs, the metabolic activity is higher compared to group 1. The third group in Figure 2 includes the last OPE pair, BBOEP/TBOEP, and the two FM pairs, TBBA/EHTBB and TBMEHP/BEHTBP. For these pairs, the parent compound was not detected in the eggs, resulting in an abundance of 100% of the corresponding metabolite. TBOEP was below the limits of detection in all samples, while BBOEP was present in all 21 eggs. An explanation for this could be that TBOEP may have undergone rapid metabolic transformation in the eagles and is therefore only represented by its metabolite. However, the instrument detection limit for TBOEP via GC-MS analysis is high (50 ng), so it is likely that the absence of TBOEP is an artifact of instrumental analytical deficiency.31 This is further supported by the relatively high levels reported in studies that utilized LC-MS/MS.12,19 Similarly, the parent FM compounds EHTBB and BEHTBP were not detected in the eggs, suggesting that rapid metabolic transformation may have occurred for these compounds. BEHTBP is the brominated analogue of bis(2-ethylhexyl) phthalate; phthalates have been reported to be easily and rapidly metabolized, usually excreted in less than 24 h and subsequently low bioaccumulation potential.32 The presence of these degradation products in the eggs indicate that OPEs and FM components can be metabolized by bald eagles at various rates depending on the specific

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REFERENCES

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DOI: 10.1021/acs.estlett.8b00163 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX