Microbial O-methylation of the flame retardant tetrabromobisphenol-A

Article abstract of DOI:10.1021/es800038q

We demonstrated the O-methylation of tetrabromobisphenol-A (TBBPA) [4,4′-isopropylidenebis (2,6-dibromophenol)] to its mono- and dimethyl ether derivatives by microorganisms present in different sediments. A most probable number assay of a marsh sediment suggested that up to 10% of the total aerobic heterotrophs may be capable of O-methylation. Although TBBPA dimethyl ether is not produced in industry, it has been detected in terrestrial and aquatic sediments. Our study supports the hypothesis that TBBPA dimethyl ether is a product of microbial O-methylation. The O-methylation of TBBPA, as well as its analog, tetrachlorobisphenol-A (TCBPA), was also demonstrated in cultures of two chlorophenol-metabolizing bacteria, Mycobacterium fortuitum CG-2 and Mycobacterium chlorophenolicum PCP-I. These strains also mediated the O-methylation of 2,6-dibromophenol and 2,6-dichlorophenol, analogs of TBBPA and TCBPA, to their corresponding anisoles, but 2,6-fluorophenol was not transformed. Due to the addition of hydrophobic methyl groups, O-methylated derivatives are more lipophilic, increasing the probability of bioaccumulation in the food chain. Future research regarding the toxicological effects of the O-methylated derivatives of TBBPA is recommended and will further elucidate potential risks to environmental and human health.

Full text of DOI:10.1021/es800038q

Environ. Sci. Technol. 2008, 42, 5555–5561
workers, laboratory personnel, and the general population
Microbial O-Methylation of the
Flame Retardant
(10–12). It is unclear whether such exposure to the general
population is the result of direct contact with TBBPA-
containing products or indirect exposure to environmental
contamination from terrestrial or atmospheric sources.
Regardless of this uncertainty, the rising global market
demand for TBBPA unfortunately ensures increasing con-
tamination in the future. There is thus a heightened concern
regarding the environmental fate and human exposure to
TBBPA and other brominated flame retardants.
At high concentrations, TBBPA is acutely toxic to a variety
of organisms (9). In particular, aquatic organisms such as
fish, algae, and crustaceans are susceptible to TBBPA-induced
toxicity. Evidence for low-dose toxicity is limited, although
in vitro assays have linked TBBPA exposure to free radical
production (13), cell death (14), and inhibition of neu-
rotransmitter transport (15). Numerous studies have impli-
cated TBBPA as a disruptor of thyroid and reproductive
hormone function (16–19). Although knowledge of TBBPA
toxicology is increasing, our understanding is still quite
limited and many reports in the literature are conflicting
(see ref (8)).
Given the widespread use of TBBPA in computers, plastics,
electronics, and other consumer products, continued de-
mand for the compound is expected. With the increasing
use of TBBPA comes a heightened need to elucidate its
environmental fate. Microorganisms are the major mediators
for the cycling of halogenated organic compounds in the
environment (20). It is therefore essential to fully delineate
and understand the diverse microbial roles in biodegradation
and biotransformation processes. Laboratory and field studies
indicate that TBBPA degrades very slowly in the environment.
Biodegradation of TBBPA under aerobic conditions has not
been demonstrated. TBBPA is, however, reductively deha-
logenated under anaerobic conditions to yield bisphenol A
(21–26). Although bisphenol A is biodegradable under aerobic
conditions (24, 27–29) it is highly recalcitrant in anaerobic
sediments (21).
Biotransformation, an alternative to degradation, alters
the compound without making substantial changes to the
carbon skeleton of the substrate. Microbial O-methylation
is one such biotransformation reaction commonly observed
for many halogenated phenolic compounds (30–32). The
microbial biotransformation products may differ greatly in
their chemical characteristics (e.g., water solubility, parti-
tioning onto solids) and more importantly their bioaccu-
mulation potential and toxicity. Although some data exist on
the accumulation and toxicology of TBBPA, little effort has
been made to analyze potential metabolites that may exist
in natural ecosystems. We thus set out to determine whether
TBBPA and its analogs may be O-methylated in contaminated
sediments and soils. Our study demonstrates that O-
methylating microorganisms appear to be prevalent in these
environments. O-Methylation of TBBPA and TCBPA and
halogenated phenol analogs was also demonstrated with two
soil bacteria, Mycobacterium fortuitum CG-2 and Mycobac-
terium chlorophenolicum PCP-1, previously shown to O-
methylate chlorinated phenolics (33). Discerning the possible
microbial metabolites of TBBPA, as well as the prevalence
of the O-methylation reaction, is important for evaluating
the environmental fate and health effects of brominated flame
retardants.
Tetrabromobisphenol-A
K E V I N W . G E O R G E A N D
¨
M A X M . H A G G B L O M *
Department of Biochemistry and Microbiology, School of
Environmental and Biological Sciences, Rutgers, The State
University of New Jersey, 76 Lipman Drive, New Brunswick,
New Jersey 08901-8525
Received January 6, 2008. Revised manuscript received
April 23, 2008. Accepted May 2, 2008.
We demonstrated the O-methylation of tetrabromobisphenol-A
(TBBPA) [4,4′-isopropylidenebis (2,6-dibromophenol)] to its
mono- and dimethyl ether derivatives by microorganisms present
in different sediments. A most probable number assay of a
marsh sediment suggested that up to 10% of the total aerobic
heterotrophs may be capable of O-methylation. Although
TBBPA dimethyl ether is not produced in industry, it has been
detected in terrestrial and aquatic sediments. Our study
supports the hypothesis that TBBPA dimethyl ether is a product
of microbial O-methylation. The O-methylation of TBBPA, as
well as its analog, tetrachlorobisphenol-A (TCBPA), was also
demonstrated in cultures of two chlorophenol-metabolizing
bacteria, Mycobacterium fortuitum CG-2 and Mycobacterium
chlorophenolicum PCP-1. These strains also mediated the
O-methylation of 2,6-dibromophenol and 2,6-dichlorophenol,
analogs of TBBPA and TCBPA, to their corresponding anisoles,
but 2,6-fluorophenol was not transformed. Due to the addition
of hydrophobic methyl groups, O-methylated derivatives are
more lipophilic, increasing the probability of bioaccumulation in
the food chain. Future research regarding the toxicological
effectsoftheO-methylatedderivativesofTBBPAisrecommended
and will further elucidate potential risks to environmental
and human health.
Introduction
Brominated flame-retardants are used extensively as additives
for fire prevention in a wide range of consumer products,
including plastics, textiles, and electronics (1). The most
common brominated flame retardant, tetrabromobisphe-
nol-A (TBBPA) [4,4′-isopropylidenebis (2,6-dibromophenol)],
with an annual global market in the range of over 130 million
kg in 2002 and over 170 million kg in 2004 (2), is predomi-
nantly used in the production of circuit boards. Because of
their widespread use, brominated flame retardants have
disseminated worldwide as environmental contaminants.
TBBPA is recalcitrant and has been detected in soils and
sediments around the world (1, 3–7).
TBBPA has been detected in aquatic and terrestrial biota
in multiple studies (see refs 3 and 8 for reviews). Although
the potential for TBBPA volatilization is low (9), human
exposure to TBBPA is reflected in the detection of TBBPA in
serum from computer technicians, electronic assembly
Experimental Procedures
Substrates. The substrates utilized in this study were
tetrabromobisphenol-A (TBBPA), tetrachlorobisphenol-A
* Corresponding author phone: (732) 932-9763ext. 326; fax: (732)
932-8965; e-mail: haggblom@aesop.rutgers.edu.
9
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 2008 American Chemical Society
VOL. 42, NO. 15, 2008
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ENVIRONMENTAL SCIENCE & TECHNOLOGY 5555
Published on Web 06/14/2008

(TCBPA), 2,6-dibromophenol (DBP), 2,6-dichlorophenol
(DCP), 2,6-difluorophenol (DFP), and tetrabromobisphenol-A
dimethyl ether. With the exception of the dimethyl ether, the
compounds were produced by Sigma Aldrich and of >98%
purity.
increased by 20 °C min-1 to a final temperature of 280 °C.
Substrates and metabolites were identified through mass
spectrometry equipped to scan from 35 to 600 m/z. The
relative abundance of 2,6-DBP, 2,6-DCP, and 2,6-DFP (as
acetyl derivatives) and their O-methylated metabolites was
quantified from the total ion chromatograms. To quantify
the relative abundance of TBBPA and its metabolites, selected
ion monitoring (SIM) of key mass fragments was utilized:
529 (TBBPA), 543 (TBBPA monomethyl ether), and 557
(TBBPA dimethyl ether). The abundances of TBBPA and
metabolites were determined from the SIM abundance
corrected for the relative abundance of the selected ion in
the total mass spectrum of each compound.
Synthesis of TBBPA Dimethyl Ether. TBBPA (in acetone)
was reacted with excess methyl iodide (CH3I) and potassium
carbonate (K2CO3) under reflux conditions following the
method of Tulp (34). After reflux the reaction yield was
determined by gas chromatography-mass spectrometry
(GC-MS; see Analysis section). The acetone solution was
decanted into a vial and evaporated to dryness, yielding
TBBPA dimethyl ether crystals of >99% purity as determined
by GC-MS. A stock solution of TBBPA dimethyl ether was
prepared in acetone and utilized for further experiments.
Culture Conditions. Mycobacterium chlorophenolicum
strain PCP-1 and Mycobacterium fortuitum strain CG-2 were
originally isolated from chlorophenol-contaminated soil and
sludge (33, 35, 36). The bacteria were grown in tryptic soy
broth (30 g L-1) at 28 °C under gentle agitation for 5 days.
The cells were collected by centrifugation and resuspended
to a density of 108 to 109 cells mL-1 in a basic mineral salts
medium supplemented with 0.1% glucose as a carbon source.
The mineral salts medium was composed of (in g L-1): 2.9
K2HPO4, 2.1 KH2PO4, 2.0 NH4Cl, 0.4 MgSO4•7H2O, 0.03 NaCl,
0.003 CaCl2, and 0.001 FeSO4•7H2O. Halogenated substrates
were added to a series of tubes from acetone stocks to give
a final concentration of 50 µM. The acetone was evaporated
and the tubes were inoculated with 2 mL of culture media.
At various time points, these tubes were sacrificed and
analyzed by GC-MS for metabolite formation.
Establishment of Sediment Microcosms. Sediments for
two different sites, Kearny Marsh, NJ (collected in July 2004)
and the Kymijoki River, Finland (collected in 2001) were
stored at 4 °C until used for preparation of aerobic micro-
cosms. Prior to setup, an acetone stock of TBBPA was added
to the microcosm flasks to give a final nominal concentration
of 10 µM and evaporated to dryness, leaving a coating of
TBBPA on the vial. The microcosms were then filled with a
slurry of 20% sediment and 80% distilled H2O (1 mL
sediment/4 mL water) and monitored for O-methylation over
time.
Results
Transformation of TBBPA and Production of Two Me-
tabolites Mediated by Mycobacterium Strains. Since a
variety of bacteria and fungi have previously been shown to
O-methylate chlorinated phenols, we wanted to determine
whether they may also mediate the transformation of the
phenolic flame retardant, TBBPA. We therefore tested the
ability of two Mycobacterium strains, CG-2 and PCP-1, to
O-methylate TBBPA.
TBBPA was added to dense cultures (108 to 109 cells mL-1
)
at a concentration of 50 µM. GC-MS analysis of cultures of
Mycobacterium fortuitum CG-2 indicated the complete
removal of TBBPA after 10 days. This loss of TBBPA correlated
with the production of increasing concentrations of two
distinct metabolites. A gas chromatogram of TBBPA and its
metabolites, as well as mass spectra identifications, is shown
in Figure 1a. Based on the bromine isotope pattern and key
mass fragments, the metabolites were identified as the methyl
and dimethyl ether derivatives of TBBPA and were identical
to the chemically synthesized derivatives. The biotransfor-
mation of TBBPA with production of TBBPA mono- and
dimethyl ethers by M. fortuitum strain CG-2 is shown in
Figure 2a. Heat-killed controls showed no TBBPA loss or
metabolite production, implicating the bacterial strain as
the catalyst for O-methylation.
Cultures of Mycobacterium chlorophenolicum PCP-1 also
mediated the transformation of TBBPA coupled to metabolite
production but at a slower rate (Figure 2c). Mass spectral
analysis again identified the two metabolites as TBBPA methyl
and dimethyl ether, respectively.
Bacterial Mediated O-Methylation of TCBPA. TCBPA,
the chlorine substituted analog of TBBPA, was similarly
transformed by cultures of Mycobacterium fortuitum CG-2
and Mycobacterium chlorophenolicum PCP-1. The loss of
TCBPA correlated to the production of two metabolites
identified through mass spectral analysis of key fragments
as TCBPA mono- and dimethyl ether, respectively (Figure
1b).
Strains CG-2 and PCP-1 O-methylated 50 µM TCBPA at
a rate similar to that observed for TBBPA. After 10 days,
Mycobacterium fortuitum CG-2 mediated the complete
conversion of TCBPA into TCBPA monomethyl and dimethyl
ether (Figure 2b). Mycobacterium chlorophenolicum PCP-1
also initiated the transformation of TCBPA into its methyl-
ether derivatives (Figure 2d). No transformation of TCBPA
was observed in sterile controls confirming the microbio-
logical basis for the O-methylation reaction.
Effect of Halogen Substituents on O-Methylation. To
determine the effect of steric changes of the halogen
substituent on the rate of O-methylation, the TBBPA analogs
2,6-DBP, 2,6-DCP, and 2,6-DFP were tested with the two
O-methylating Mycobacterium strains. M. fortuitum strain
CG-2 mediated the transformation of 2,6-DBP coupled with
the accumulation of a metabolite (Figure 3a) identified by
mass spectral analysis as 2,6-dibromoanisole, the methylated
derivative of 2,6-DBP. An identical conversion of 2,6-DCP
MPN Assay. The number of O-methylating microbes g-1
sediment was determined for the Kearny Marsh sediment
using a most probable number (MPN) assay (37, 38). A five
replicate MPN series from 10-1 to 10-9 was prepared. Each
tube contained TBBPA (20 µM) and 2 mL of diluted sediment
in mineral salts media (see composition above) containing
0.1% yeast extract as a complex carbon source. After
incubation at 28 °C for 45 days, the tubes with visual turbidity
were scored positive for heterotrophs (total number of
heterotrophs). The tubes were then extracted with 1 mL of
hexane and analyzed for O-methylation using GC-MS. Based
on detection limits for TBBPA and O-methyl derivatives and
background signal in negative controls any conversion >1.0%
of TBBPA to methylated forms was scored as positive for
O-methylation. The most probable number of total het-
erotrophs and O-methylating microorganisms was then
estimated using the most probable number calculator of Klee
(39).
Analysis. Samples for analysis of TBBPA, TCBPA, and
O-methyl derivatives by gas chromatography-mass spec-
trometry were extracted in hexane (1:1) for 1 h. The other
substrates, 2,6-DBP, 2,6-DCP, and 2,6-DFP, were first acety-
lated with acetic anhydride in a carbonate buffer prior to
extraction as described previously (33). These samples were
analyzed on an Agilent HP 6890 GC-MS equipped with an
HP-5MS column (30 m × 250 µm i.d., nominal film thickness
0.25 µm). The carrier gas was helium at a flow of 1 mL min-1
.
The temperature ramp began at 60 °C for 1 min, then
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5556

FIGURE 1. Gas chromatography and mass spectra of TBBPA (a) and TCBPA (b) and their O-methylated metabolites.
into2,6-dichloroanisolewasobserved(Figure3b).M.cholorophe-
nolicum strain PCP-1 exhibited similar behavior, O-methy-
lating both 2,6-DBP and 2,6-DCP to their corresponding
anisoles (Figure 3d and e). The fluorinated derivative, 2,6-
DFP, was not O-methylated by either strain (Figure 3c and
f). No methylation of 2,6-DBP, 2,6-DCP, and 2,6-DFP was
observed in sterile controls (data not shown).
O-Methylation of TBBPA in Sediment Microcosms. To
determine the potential for microbial TBBPA O-methy-
lation in the environment, microcosms were established
using sediment from Kearny Marsh, New Jersey, and
Kymijoki River, Finland and spiked with a TBBPA con-
centration of 10 µM. After 80 days, over 50% of the original
TBBPA was transformed to TBBPA mono- and dimethyl
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FIGURE 2. Conversion of TBBPA (a, c) and TCBPA (b, d) to mono- and dimethylated forms by M. fortuitum strain CG-2 (top) and M.
chlorophenolicum strain PCP-1 (bottom). Data points are means of triplicate cultures ( one standard deviation.
FIGURE 3. O-Methylation of 2,6-DBP (a, d), 2,6-DCP (b, e), and 2,6-DFP (c, f) to their corresponding anisoles by M. fortuitum strain
CG-2 (top) and M. chlorophenolicum strain PCP-1 (bottom). Data points are means of triplicate cultures ( one standard deviation.
ether in the Kearny Marsh sediment (Figure 4a). Heat-
killed, sterile controls showed no transformation over the
80-day period. After 60 days, approximately 10% of the
TBBPA was converted to mono- and dimethylated deriva-
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FIGURE 5. Sequential O-methylation of TBBPA to mono- and
dimethylated derivatives.
for their ability to O-methylate chlorophenols transform both
TBBPA and TCBPA to the corresponding mono- and dim-
ethylated ethers (Figure 5). Additionally this study has
demonstrated the microbially mediated O-methylation of
TBBPA in sediment microcosms. This suggests that O-
methylation of TBBPA may be an environmentally significant
process.
Our data from Kearny Marsh suggest that TBBPA O-
methylating microorganisms may be prevalent in the en-
vironment. In this sediment, approximately 10% of the total
aerobic heterotrophs were capable of O-methylation. It thus
seems likely that O-methylation will be a prominent reaction
in contaminated sediments. Furthermore, previous studies
have shown that a variety of bacteria and fungi mediate
O-methylation of halogenated phenols (see ref (31) for
review). In addition to TBBPA, numerous other halogenated
anisoles of no known anthropogenic source have been
detected in environmental and biological samples (41–45).
The ubiquity of O-methylating microorganisms suggests that
these compounds may also be products of biological O-
methylation.
In the two Mycobacterium strains tested here, there
seemed to be little difference in the rates of methylation of
brominated versus chlorinated phenolics. It was previously
demonstrated that the O-methylating enzyme(s) of many
methylating bacteria prefer substrates with the hydroxyl
group flanked by two halogen substituents, usually chlorine
or bromine (30, 32, 46). For example, Rhodococcus strains
did not O-methylate 2-chlorophenol or phenol (30). This
preference for two bulky flanking substituents suggests a
possible explanation for the lack of methylation of 2,6-DFP.
Since the fluorine ion is the smallest of the halogens, the
flanking substituents are likely too close in size to hydrogen
to be recognized by the enzyme. The van der Waals radius
of fluorine is 1.35 Å, quite similar to hydrogen’s radius of
1.20 Å. The methyltransferase enzyme’s suspected geometric
selectivity also explains the similar methylation rates of 2,6-
DBP and 2,6-DCP as the van der Waals radii of Br and Cl are
1.80 Å and 1.95 Å, respectively (47).
FIGURE 4. O-Methylation of TBBPA in Kearny Marsh sediment
(a) and Kymijoki sediment (b) cultures. Data points are means
of triplicate cultures ( one standard deviation.
tives in Kymijoki cultures (Figure 4b). Sterile controls
showed no activity.
Enumeration of O-Methylating Microorgansism in
Kearny Marsh Sediment. A 5-tube MPN assay was performed
to estimate the abundance of O-methylating organisms in
the Kearny Marsh sediment. The MPN test for O-methylation
gave an estimate of 1.3 × 106 methylators g-1 soil (95%
confidence limit 4.3 × 105 to 4.1 × 106). The total number
of heterotrophs was estimated to be 1.1 × 107 g-1 soil (95%
confidence limit 2.8 × 106 to 3.3 × 107), suggesting that up
to 10% of the heterotrophs in the Kearny Marsh sediment
may be capable of O-methylation.
Discussion
The abundant use of TBBPA and other brominated flame
retardants has led to increasing environmental contamina-
tion. Although TBBPA is resistant to aerobic and anaerobic
degradation, transformation reactions, such as reductive
debromination and O-methylation, alter the compound
without making changes to the carbon skeleton of the
substrate and can be an alternative to degradation. Envi-
ronmental and toxicological data concerning potential
products of this process are sparse (9). Although TBBPA
dimethyl ether is not produced in industry, it has been
detected in samples of terrestrial and aquatic sediments, as
well as biological samples (5, 7, 40). In these studies, it was
hypothesized that the TBBPA dimethyl ether may be a product
of microbial transformation. Allard et al. (32) first demon-
strated that bacterial cultures were capable of O-methylating
TBBPA although the reaction proceeded at a relatively slow
rate. We show here that two Mycobacterium isolates known
While we have established that TBBPA may be trans-
formed under aerobic conditions to TBBPA mono- and
dimethyl ether, there is no information on their fate in either
aerobic or anaerobic environments. With the addition of two
hydrophobic methyl groups, TBBPA dimethyl ether is more
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lipophilic than its parent compound. This characteristic
increases its potential for bioconcentration in fatty tissue.
The presence of TBBPA dimethyl ether has been demon-
strated in aquatic organisms such as fish and mussels in
Japan (7, 48), as well as peregrine Falcon eggs in Greenland
(49). A dimethoxylated polybrominated biphenyl, a com-
pound similar in structure to TBBPA dimethyl ether, has been
detected in the blubber of various marine mammals off the
coast of Japan (43). Currently, little is known about the
toxicology of TBBPA mono- and dimethylated ether. Given
the suspected prevalence of bacterial O-methylation, ad-
ditional environmental and toxicological data should be
collected for these derivatives.
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Acknowledgments
K.G. was the recipient of a Rutgers University Undergraduate
Research Fellowship. This work was supported in part
through the Strategic Environmental Research and Develop-
ment Program (CU-1208 and ER-1492).
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