David M.V. Saunders

In vitro endocrine disruption and TCDD-like effects of three novel brominated flame retardants: TBPH, TBB, & TBCO

The novel brominated flame retardants (NBFRs), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), Bis(2-ethylhexyl)-2,3,4,5-tetrabromophtalate (TBPH), and 1,2,5,6-tetrabromocyclooctane (TBCO) are components of flame retardant mixtures including Firemaster 550 and Saytex BC-48. Despite the detection of these NBFRs in environmental and biotic matrices, studies regarding their toxicological effects are poorly represented in the literature. The present study examined endocrine disruption by these three NBFRs using the yeast YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay. The NBFRs produced no TCDD-like effects in the H4IIE assay or agonistic effects in the YES/YAS assays. TBB produced a maximal antiestrogenic effect of 62% at 0.5mgL(-1) in the YES assay while TBPH and TBCO produced maximal antiandrogenic effects of 74% and 59% at 300mgL(-1) and 1500mgL(-1), respectively, in the YAS assay. Significant effects were also observed in the H295R assay. At 0.05mgL(-1), 15mgL(-1), and 15mgL(-1) TBB, TBPH, and TBCO exposures, respectively resulted in a 2.8-fold, 5.4-fold, and 3.3-fold increase in concentrations of E2. This is one of the first studies to demonstrate the in vitro endocrine disrupting potentials of TBB, TBPH, and TBCO.

Effects of tris (2-butoxyethyl) phosphate (TBOEP) on endocrine axes during development of early life stages of zebrafish (Danio rerio)

Due to phasing out of additive flame retardants such as polybrominated diphenyl ethers (PBDEs), Tris (2-butoxyethyl) phosphate (TBOEP) is widely used as a substitute. TBOEP is ubiquitous in the environment and has been measured at concentrations of micrograms per liter (μg L(-1)) in surface waters and wastewater. Information on potential adverse effects on development of aquatic organisms caused by exposure to environmentally relevant concentrations of TBOEP is limited, especially for effects that may be caused through impairment of endocrine-modulated homeostasis. Therefore, this study was conducted to determine effects of TBOEP on ontogeny and transcription profiles of genes along the hypothalamus-pituitary-thyroidal (HPT), hypothalamus-pituitary-adrenal (HPA), and hypothalamus-pituitary-gonadal (HPG) axes in embryos/larvae of zebrafish (Danio rerio). Exposure to TBOEP (2-5,000 μg L(-1)) from 3 h post-fertilization (hpf) to 120 hpf induced developmental malformations in zebrafish with a LC50 of 288.54 μg L(-1) at both 96 hpf and 120 hpf. The predicted no observed effect concentration (PNOEC) was 2.40 μg L(-1). Exposure to 2, 20, or 200 μg TBOEP L(-1) altered expression of genes involved in three major molecular pathways in a concentration-dependent manner after 120 hpf. TBOEP caused lesser expression of some genes involved in synthesis of hormones, such as (pomc and fshβ) as well as upregulating expression of some genes coding for receptors (thr, tshr, gr, mr, er and ar) in zebrafish larvae. These changes at the molecular level could result in alterations of endocrine function, which could result in edema or deformity and ultimately death.

Untargeted Identification of Organo-Bromine Compounds in Lake Sediments by Ultrahigh-Resolution Mass Spectrometry with the Data-Independent Precursor Isolation and Characteristic Fragment Method

While previous studies have found that unknown natural and synthetic organo-bromine compounds (NSOBCs) contributed more than 99% of the total organic bromine (Br) in the environment, there was no efficient method for untargeted screening to identify NSOBCs in environmental matrixes. A novel untargeted method for identifying NSOBCs, based on ultrahigh-resolution mass spectrometry (UHRMS) with the Q Exactive instrument was developed. This method included a data-independent precursor isolation and characteristic fragment (DIPIC-Frag) procedure to identify NSOBCs. A total of 180 successive 5-m/z-wide windows were used to isolate precursor ions. This resulted in a sufficient dynamic range and specificity to identify peaks of Br fragment ions for analysis. A total of 2520 peaks of NSOBC compounds containing Br were observed in sediments from Lake Michigan, United States. A new chemometric strategy which combined chromatographic profiles, isotopic peaks, precursor isolation window information, and intensities was used to identify precursor ions and chemical formulas for detecting NSOBCs. Precursor ions for 2163 of the 2520 NSOBCs peaks (86%) were identified, and chemical formulas for 2071 NSOBCs peaks (82%) were determined. After exclusion of isotopic peaks, 1593 unique NSOBCs were identified and chemical formulas derived for each. Most of the compounds identified had not been reported previously and had intensities which were 100- to 1000-fold greater than the congeners of polybrominated diphenyl ethers (PBDEs). In extracts of sediments, these compounds exhibited variations in intensities (<10(3) to ∼10(8)), m/z values (170.9438-997.5217), retention times on a C18 column (1.0-29.3 min), and the number of Br atoms (1-8). Generally, compounds with greater m/z values had longer retention times and greater numbers of Br atoms. Three compounds were used in a proof-of-concept experiment to demonstrate that structures of some of the screened NSOBCs could be further predicted by combining searching of database libraries and high-resolution MS(2) spectra.

Untargeted Screening and Distribution of Organo-Bromine Compounds in Sediments of Lake Michigan

Previously unreported natural and synthetic organo-bromine compounds (NSOBCs) have been found to contribute more than 99% of total organic bromine (TOB) in environmental matrices. We recently developed a novel untargeted method (data-independent precursor isolation and characteristic fragment, DIPIC-Frag) and identified ∼2000 NSOBCs in two sediments from Lake Michigan. In this study, this method was used to investigate the distributions of these NSOBCs in 23 surficial samples and 24 segments of a sediment core from Lake Michigan. NSOBCs were detected in all 23 surficial samples and exhibited 10- to 100-fold variations in peak abundance among locations. The pattern of distributions of NSOBCs was correlated with depth of the water column (r(2) = 0.61, p < 0.001). Hierarchical cluster analysis showed that sediments in close proximity exhibited similar profiles of NSOBCs. Distributions of NSOBCs in 24 segments of a sediment core dated from 1766 to 2008 were investigated, and samples from similar depths exhibited similar profiles of NSOBCs. NSOBCs were grouped into four clusters (soft-cluster analysis) with different temporal trends of abundances. 515 and 768 of the NSOBCs were grouped into cluster 1 and cluster 3 with increasing temporal trends, especially since 1950, indicating that abundances of these compounds might have been affected by human activities.

Detection, Identification, and Quantification of Hydroxylated Bis(2-ethylhexyl)-Tetrabromophthalate Isomers in House Dust

Ultra-High Resolution LC/mass spectrometry (LC-UHRMS; Thermo Fisher Q-Exactive) was used to identify two novel isomers of hydroxylated bis(2-ethylhexyl)-tetrabromophthalate (OH-TBPH) which were unexpectedly observed in a commercial standard of TBPH. By combining ultra-high resolution (UHR) mass spectra (MS(1)), mass errors to theoretical [TBPH-Br+O](-) were 2.1 and 1.0 ppm for the two isomers, UHR-MS(2) spectra and NMR analysis; the structures of the two compounds were identified as hydroxylated TBPH with a hydroxyl group on the aromatic ring. Relatively great proportions of the two isomers of OH-TBPH were detected in two technical products, Firemaster 550 (FM-550; 0.1% and 6.2%, respectively) and Firemaster BZ 54 (BZ-54; 0.1% and 7.9%), compared to a commercial standard (0.4% and 0.9%). To simultaneously analyze OH-TBPH isomers and TBPH in samples of dust, a method based on LC-UHRMS was developed to quantify the two compounds, using negative and positive ion modes, respectively. The instrumental limit of detection for TBPH was 0.01 μg/L, which was 200-300 times better than traditional methods (2.5 μg/L) based on gas chromatography-mass spectrometry. The analytical method combined with a Florisil cleanup was successfully applied to analyze TBPH and OH-TBPH in 23 indoor dust samples from Saskatoon, Saskatchewan, Canada. Two OH-TBPH isomers, OH-TBPH1 and OH-TBPH2, were detected in 52% and 91% of dust samples, respectively. Concentrations of OH-TBPH2 (0.35 ± 1.0 ng/g) were 10-fold greater than those of OH-TBPH1 (0.04 ± 0.88 ng/g) in dust, which was similar to profiles in FM-550 and BZ-54. TBPH was also detected in 100% of dust samples with a mean concentration of 733 ± 0.87 ng/g. A significant (p < 0.001) log-linear relationship was observed between TBPH and OH-TBPH isomers, further supporting the hypothesis of a common source of emission. Relatively small proportions of OH-TBPH isomers were detected in dust (0.01% ± 0.67 OH-TBPH1 and 0.1% ± 0.60 OH-TBPH2), which were significantly less than those in technical products (p < 0.001). This result indicated different environmental behaviors of OH-TBPH and TBPH. Detection of isomers of OH-TBPH is important, since compounds with phenolic groups have often shown relatively greater toxicities than nonhydroxylated analogues. Further study is warranted to clarify the environmental behaviors and potential toxicities of OH-TBPH isomers.

Inhibition of ABC transport proteins by oil sands process affected water

The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI(+) and ESI(-) mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O(+), SO(+), and NO(+) chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2(-)), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates this effect and inhibition of the activity of MRPs by OSPW from Base Mine Lake does not occur at environmentally relevantconcentrations.

A mixture of the novel brominated flame retardants TBPH and TBB affects fecundity and transcript profiles of the HPGL-axis in Japanese medaka.

The novel brominated flame retardants (NBFRs), bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) and 2-ethylhexyl-2,3,4,5 tetrabromobenzoate (TBB) are components of the flame retardant mixture Firemaster 550 and both TBPH and TBB have recently been listed as high production volume chemicals by the US EPA. These NBFRs have been detected in several environmental matrices but very little is known about their toxic effects or potencies. Results of in vitro assays demonstrated potentials of these NBFRs to modulate endocrine function through interactions with estrogen (ER) and androgen receptors (AR) and via alterations to synthesis of 17-β-estradiol (E2) and testosterone (T), but in vivo effects of these chemicals on organisms are not known. Therefore a 21-day short term fish fecundity assay with Japanese medaka (Oryzias latipes) was conducted to investigate if these NBFRs affect endocrine function in vivo. Medaka were fed a diet containing either 1422 TBPH:1474 TBB or 138:144 μg/g food, wet weight (w/w). Cumulative production of eggs was used as a measure of fecundity and abundances of transcripts of 34 genes along the hypothalamus-pituitary-gonadal-liver (HPGL) axis were quantified to determine mechanisms of observed effects. Cumulative fecundity was impaired by 32% in medaka exposed to the greatest dose of the mixture of TBPH/TBB. A pattern of global down-regulation of gene transcription at all levels of the HPGL axis was observed, but effects were sex-specific. In female medaka the abundance of transcripts of ERβ was lesser in livers, while abundances of transcripts of VTG II and CHG H were greater. In male medaka, abundances of transcripts of ERα, ERβ, and ARα were lesser in gonads and abundances of transcripts of ERβ and ARα were lesser in brain. Abundances of transcripts of genes encoding proteins for synthesis of cholesterol (HMGR), transport of cholesterol (HDLR), and sex hormone steroidogenesis (CYP 17 and 3β-HSD) were significantly lesser in male medaka, which might have implications for concentrations of sex hormones. The results of this study demonstrate that exposure to components of the flame retardant mixture Firemaster(®) 550 has the potential to impair the reproductive axis of fishes.

Untargeted Screening and Distribution of Organo-Iodine Compounds in Sediments from Lake Michigan and the Arctic Ocean

The majority of halogenated organic compounds present in the environment remain unidentified. To address this data gap, we recently developed an untargeted method (data-independent precursor isolation and characteristic fragment; DIPIC-Frag) for identification of unknown organo-bromine compounds. In this study, the method was adapted to enable untargeted screening of natural and synthetic organo-iodine compounds (NSOICs) in sediments. A total of 4,238 NSOIC peaks were detected in sediments from Lake Michigan. Precursor ions and formulas were determined for 2,991 (71%) of the NSOIC peaks. These compounds exhibited variations in abundances (<10(3) to ∼10(7)), m/z values (206.9304-996.9474), retention times (1.0-29.7 min), and number of iodine atoms (1-4). Hierarchical cluster analysis showed that sediments in closer proximity exhibited similar profiles of NSOICs. NSOICs were screened in 10 samples of sediment from the Arctic Ocean to compare the profiles of NSOICs between freshwater and marine sediments. A total of 3,168 NSOIC peaks were detected, and profiles of NSOICs in marine sediments were clearly distinct from Lake Michigan. The coexistence of brominated and iodinated analogues indicated that some NSOICs are of natural origin. Different ratios of abundances of iodinated compounds to brominated analogues were observed and proposed as a marker to distinguish sources of NSOICs.

Effects of the brominated flame retardant TBCO on fecundity and profiles of transcripts of the HPGL-axis in Japanese medaka

The novel brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO) is an additive flame retardant which is marketed under the trade name Saytex BCL-48. TBCO has recently been investigated as a potential alternative to the major use brominated flame retardant, hexabromocyclododecane (HBCD), which could have major implications for significant increases in amounts of TBCO used. Yet there is a lack of information regarding potential toxicities of TBCO. Recently, results of in vitro experiments have demonstrated the potential of TBCO to modulate endocrine function through interaction with estrogen and androgen receptors and via alterations to the synthesis of 17-β-estradiol and testosterone. Further research is required to determine potential endocrine disrupting effects of TBCO in vivo. In this experiment a 21-day fecundity assay with Japanese medaka (Oryzias latipes) was conducted to examine endocrine disrupting effects of TBCO in vivo. Medaka were fed a diet containing either 607 or 58μg TBCO/g food, wet mass (wm). Fecundity, measured as cumulative deposition of eggs and fertilization of eggs, as well as abundances of transcripts of 34 genes along the hypothalamus-pituitary-gonadal-liver (HPGL) axis were measured as indicators of holistic endocrine disruption and to determine mechanisms of effects, respectively. Cumulative fecundity was 18% lesser by medaka exposed to 58μg TBCO/g, wm food. However, fecundity of medaka exposed to 607μg TBCO/g, wm food was not significantly different from that of controls. Organ-specific and dose-dependent alterations to abundances of transcripts were observed in male and female medaka. A pattern of down-regulation of expression of genes involved in steroidogenesis, metabolism of cholesterol, and regulatory feedback mechanisms was observed in gonads from male and female medaka which had been exposed to the greater concentration of TBCO. However, these effects on expression of genes were not manifested in effects on fertilization of eggs or fecundity. In livers from male and female medaka exposed to the lesser concentration of TBCO greater expression of genes that respond to exposure to estrogens, including vitellogenin II, choriogenin H, and ERα, were observed. The results reported here confirm the endocrine disrupting potential of TBCO and elucidate potential mechanisms of effects which include specific patterns of alterations to abundances of transcripts of genes in the gonad and liver of medaka.

Correction to Mutagenic Azo Dyes, Rather than Flame Retardants, are the Predominant Brominated Compounds in House Dust

formulas and monoisotopic m/z values, whereas this information was correctly shown in the supporting informationa. For example, the m/z for the first compound ([M+1]) should be replaced by 532.0719 [M]. A corrected Table 1 is included above. In addition, the compound formula of the first compound could not be exclusively determined between C23H23BrN3O7 and C21H21BrN6O6 with similar m/z values and potential fragments, and C21H21BrN6O6 should be the formula of the first compound, as validated by chemical standard (Disperse Blue 373). Accordantly, the compound formula of Figure 3D should be corrected from C20H18BrN3O7 to C18H16BrN6O6, and from C23H23BrN3O7 to C21H21BrN6O6. Table 1. m/z values, retention times and predicted formulas of the 10 most-abundant brominated compounds in house dust identified by DIPIC-Frag