Non-polar halogenated natural products bioaccumulated in marine samples. II. Brominated and mixed halogenated compounds

Article abstract of DOI:10.1016/S0045-6535(03)00200-5

Several identified and potential natural brominated bioaccumulative compounds were studied in this work. 4,6-dibromo-2-(2′,4′-dibromo)phenoxyanisole (BC-2) previously detected in Australian marine mammals and isolated from sponges, was synthesized. Two by

Full text of DOI:10.1016/S0045-6535(03)00200-5

Chemosphere 52 (2003) 423–431
www.elsevier.com/locate/chemosphere
Non-polar halogenated natural products bioaccumulated
in marine samples. II. Brominated and mixed
halogenated compounds
*
Walter Vetter , Wu Jun
Department of Food Chemistry, Friedrich-Schiller-University Jena, Dornburger Strasse 25, D-07743 Jena, Germany
Received 5 February 2002; received in revised form 25 April 2002; accepted 23 May 2002
Abstract
Several identified and potential natural brominated bioaccumulative compounds were studied in this work. 4,6-
dibromo-2-(20,40-dibromo)phenoxyanisole (BC-2) previously detected in Australian marine mammals and isolated from
sponges, was synthesized. Two byproducts (a tetrabromo isomer and a tribromo congener) were investigated as well.
The byproducts of the synthesis were not identified in the environmental samples investigated. Previously described
natural brominated compounds (BC-1, BC-2, BC-3, BC-10, BC-11, MHC-1) and anthropogenic brominated diphenyl
ethers (BDE-47, BDE-99, BDE-100, BDE-154) were detected in a sample of human milk. The sample was from a
woman from the Faeroe Islands who frequently consumed fish as well as whale blubber and meat. The most abundant
compound originated from the natural tetrabromo phenoxyanisole BC-3 which may have a 3:1 distribution of bromine
on the two phenyl units. This sample also accumulated a dibromochloroanisole, as well as a previously unknown mixed
halogenated compound (MHC-X) and an unknown, most likely aromatic brominated compound. Co-elutions on a DB-
5 column were found for BDE-99 and BC-11 as well as BDE-154 and the unknown brominated compound. This
suggests that quantification of these two compounds has to be carried out carefully.
Two samples of lower trophic level, namely Baltic cod liver and Mexican mussel tissue, were investigated as well. The
cod liver samples contained BDE congeners but also abundant signals for the natural 2,3,30,4,40,5,50-heptachloro-10-
methyl-1,20-bipyrrole Q1 and tribromoanisole (TBA). The mussel sample contained Q1, TBA, another halogenated
anisole, BC-1, BC-2, and BC-3, as well as additional, potential natural brominated compounds in the elution range of
tribromophenoxyanisoles.
Ó 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Naturally produced organohalogens; 4,6-dibromo-2-(20,40-dibromo)phenoxyanisole (BC-2); Synthesis; Brominated flame
retardants; Marine samples; Human milk
1. Introduction
chloropesticide––albeit this discovery was reserved to
Bender (1933)––in 1825. Bromine, however, was only
discovered the year after in 1826. This historic com-
parison appears to be typical of the scientific relevance
of the two halogens. It seems that brominated com-
pounds always lagged behind their chlorinated kins. In
the middle of the 20th century, a wide range of chlori-
nated compounds were produced at million-ton-scales
for industrial (e.g. PCBs and PCNs) and agricul-
tural (e.g. toxaphene, DDT, chlordane, and lindane)
In 1774 Schiele discovered chlorine and Faraday
synthesized hexachlorocyclohexane and thus the first
*
€
Corresponding author. Present address: Universitat Ho-
henheim, Institut fur Lebensmittelchemie (170) Garbenstrasse
€
28, D-70599 Stuttgart, Germany. Tel.: +49-711-459-4016; fax:
+49-711-459-4377.
E-mail address: w-vetter@uni-hohenheim.de (W. Vetter).
0045-6535/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0045-6535(03)00200-5

424
W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
applications. It took again some decades until the bro-
minated compounds received industrial attention. These
days, it almost appears that large amounts of bromi-
nated compounds were only produced after the ban of
the lighter halogen.
2. Materials and methods
2.1. Synthesis of BC-2
2.1.1. Synthesis of 2-methoxy-3,5-dibromophenol (Green,
1991)
TodayÕs major industrial field of application of bro-
minated compounds is for flame retardation. In past
years the records on the identification of residues of
brominated flame retardants (BFRs) in the environment
increased (Marsh et al., 1999; de Boer et al., 2000).
Consequently, it seemed that the brominated com-
pounds that bioaccumulated in marine organisms are
BFRs due to the lack of other plausible sources. From
this point of view it was thought, that compounds ap-
pearing at non-typical retention times in gas chromato-
grams might be unknown yet unidentified congeners of
BFRs. This is not true for a number of brominated
compounds we have detected in various samples (e.g.
BC-2, BC-11, MHC-1) (Vetter et al., 2001a,b). Two of
them (BC-2 and BC-11) were recently identified as natu-
rally produced organohalogens (Vetter et al., 2002b),
and the same was expected for several other (previous)
unknowns.
2,4,6-Tribromoanisole (10 g, 30 mmol) was sus-
pended in 200 ml of dry n-pentane and cooled to )20 °C
in an Ar atmosphere. Twenty milliliter of n-butyllithium
(1.6 g, 32 mmol) in n-hexane was added over 10 min with
vigorous mechanical stirring. This suspension was al-
lowed to warm to )10 °C over 15 min. Upon cooling to
)30 °C, neat trimethyl borate 3.2 g (32 mmol) was added
all at once. The solution was warmed to 0 °C over 30
min and then cooled )10 °C. A solution of 40% per-
acetic acid/acetic acid (25 ml) was added over 30 min.
Upon completion of the addition, the solution was
warmed to 0 °C over 30 min and then cooled to )10 °C,
and 25 ml of saturated aqueous NaHSO3 was added
dropwise over 30 min. Upon warming to room tem-
perature equal volumes of water and diethylether were
added. The organic layer was separated, dried over
MgSO4, and evaporated to provide a colorless oil. Yield
7.4 g (87%), b.p. 120 °C (0.24 Torr).
In order to receive more information on the structure
of brominated compounds, we suggested to monitor
additional low-mass fragment ions in GC/ECNI-
MS-SIM studies (see Section 2) (Vetter, 2001; Vetter et al.,
2002a, 2003). When methane is used as the CI gas, [Br]À
and [HBr2]À next to [Br]À qualifies aromatic organo-
bromines, [Br2]À next to [Br]À qualifies non-aromatic
organobromines, and [BrCl]À next to [Br]À qualifies
mixed halogenated non-aromatics (Fig. 1) (Vetter, 2001;
Vetter et al., 2002a). In this presentation we add more
knowledge to the current state of the art of poten-
tial natural brominated compounds bioaccumulated
in the lipids of members of marine food webs. Chlori-
nated compounds are discussed elsewhere (Vetter et al.,
2003).
2.1.2. Synthesis of 2,20,4,40-tetrabromodiphenyliodonium
chloride (III) (Marsh et al., 1999)
A mixture of concentrated sulfuric acid (2.4 ml) and
30% fumic sulfuric acid (4.8 ml) was added to iodine
(2.03 g, 8 mmol) under stirring. Then a mixture of
concentrated sulfuric acid (0.64 ml), 65% fumic sulfuric
acid (0.32 ml), and 100% fumic nitric acid (1.04 ml) was
slowly added. The reaction mixture was stirred at 70–80
°C for 1.5 h, at which time yellow crystals of iodyl
sulfate precipitated. The mixture was then cooled and
1,3-bromobenzene (10 g, 42 mmol) was slowly added at
10–15 °C. The mixture was stirred at 45 °C for 2 h and
then cooled to 0 °C. Water was carefully added in small
m/z 114
5000
m/z 116
MHC-1
5000
m/z 79
10000
20
22
24
26
28
30
32
34
36
38
40
42
[min]
Fig. 1. Identification of MHC-1 in a blubber extract of a monk seal (Monachus monachus) from northwest Africa on the basis of
fragment ions at m=z 114 and 116 (both corresponding with [BrCl]À) and m=z 79.

W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
425
portions at 5–10 °C. Nitrogen oxides were removed by
introducing a gentle stream of nitrogen into the reaction
solution. The product was collected, dissolved in meth-
anol, and crystallized as the chloride salt by dropwise
addition of concentrated hydrochloric acid. Yield 6.64 g
(62%).
programmed from 80 °C (hold time 1 min) at 20 °C/min
to 180 °C (hold time 2 min), at 2 °C/min to 200 °C (hold
time 5 min), and at 20 °C/min to 280 °C (hold time 15
min).
The transfer line, ion source and quadrupole tem-
peratures were set at 280, 150 and 100 °C, respectively.
Methane (quality 5.0; Linde, Germany) was used as the
CI moderating gas. The system was optimized by man-
ual tuning with perfluorotributylamine (PFTBA). In the
total ion current mode (TIC), m=z 50–650 were scanned.
In the selected ion monitoring (SIM) mode, the follow-
ing m=z values (corresponding compositions are given in
parentheses) were recorded in parallel after a solvent
delay of 20 min: m=z 79 ([79Br]À) and 81 ([81Br]À), m=z
158 ([79Br79Br]À), m=z 160 ([79Br81Br]À), m=z 159
([1H79Br79Br]À), m=z 161 ([1H79Br81Br]À), m=z 114
([79Br35Cl]À), m=z 116 ([79Br37Cl]À and [81Br35Cl]À), as
well as m=z 115 [1H79Br35Cl]À and m=z 117
([1H79Br37Cl]À and [1H81Br35Cl]À).
2.1.3. Synthesis of BC-2
2-Methoxy-3,5-dibromophenol 0.7 g (2.5 mmol) was
dissolved in an aqueous solution (20 ml) of sodium hy-
droxide (0.1 g, 2.5 mmol). The diphenyliodonium salt
(2.5 mmol) was added and the mixture refluxed for 1.5 h.
The product was extracted from the crude mixture using
diethylether (2 Â 30 ml). The organic layer was washed
with water and then dried over sodium sulfate. The so-
lution was filtered, the solvent was evaporated, and the
product was purified on a silica gel column with n-hex-
ane as the mobile phase. Upon re-crystallisation from
n-hexane and chromatography on 50 g silica gel, a
colourless oil was obtained which proved to be neat
BC-2.
2.3. Sample clean-up
2.2. GC/ECD and GC/MS parameters
All samples shown in this presentation were cleaned
using our validated method consisting of microwave-
assisted extraction, gel permeation chromatography,
adsorption chromatography on both deactivated and
activated silica (Vetter et al., 1998; Weichbrodt et al.,
1999, 2000). The internal standard was perdeuterated
a-HCH (a-PDHCH).
Screening on planar molecules was performed with 1
g of a 1:1 mixture of Supelclean Envicarb (120–400
mesh) and Celite 545-AW (Supelco, Deisenhofen, Ger-
many). The first fraction was eluted with 50 ml n-hexane,
and four further fractions were collected with 50 ml
toluene, respectively.
GC/ECD analyses were performed with a Hewlett-
Packard 5890 series II gas chromatograph modified by
€
Gerstel (Mulheim, Germany). A T-piece behind the PAS
split/splitless injector (splitless time 1.5 min) divided the
samples equally onto two capillary columns that ended
in 63Ni electron capture detectors (ECDs). The samples
were injected automatically (HP 7673 autosampler). The
capillary columns CP-Sil 2 and CP-Sil 8/20% C18 (both:
length 50 m, 0.25 mm i.d., 0.25 lm film thickness) were
from Chrompack (Middelburg, The Netherlands). He-
lium (5.0 quality, Institute of Physics, University of
Jena, Germany) was used as carrier gas at a constant
flow of 1.3 ml/min, and nitrogen (5.0 quality, Linde,
Germany) was used as the ECD make-up gas. Moisture
and oxygen filters from Varian-Chrompack were used.
The injector and detector temperatures were set at 250
and 300 °C, respectively. The GC oven program was the
following: after injection at 60 °C (1.5 min) the tem-
perature was ramped at 40 °C/min to 150 °C (5 min),
then ramped at 2 °C/min to 230 °C (0 min), and finally
ramped at 5 °C/min to 270 °C (15 min).
A Hewlett-Packard 5890 II plus gas chromatograph
interfaced to a 5989B MS Engine was used for electron
capture negative ion mass spectrometry (GC/ECNI-MS)
and electron impact mass spectrometry (GC/EI-MS).
Helium (see above) was used as the carrier gas. Injec-
tions into a split/splitless injector (maintained at 250 °C)
were performed in the splitless mode (1.5 min). A HP-5
(manufacturer: Hewlett-Packard/Agilent; 95% dimethyl-,
5% diphenylpolysiloxane; equivalent with DB-5 and
CP-Sil 8) capillary column (30 m, 0.25 mm i.d., 0.18 lm
film thickness) was used for analyses. The GC oven was
3. Results and discussion
The presence of halogenated compounds in top pre-
dators demands that the compounds are both lipophilic
(log Kow > 5) and persistent. Both criteria are fulfilled by
non-polar chlorinated xenobiotica such as PCBs, DDT
and other chloropesticides. Phenolic compounds are
considered as the most stable group of natural organo-
halogens (Faulkner, 1980). However, phenolic organo-
halogens have never been detected in adipose tissue of
higher organisms (Letcher et al., 2000). This suggested
that natural (halogenated) products are not bioaccu-
mulative.
On the other hand, high application rates of non-
polar BFRs (at ꢀ105 tons per year) caused increasing
pollution in the marine ecosystem upon release of the
compounds into the environment. In particular, bromi-
nated diphenyl ethers (BDEs) were repeatedly detected

426
W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
in wildlife (de Boer et al., 2000). Therefore, analysis of
brominated compounds in the environment has become
a subject for more and more scientists from all over the
world.
determination of the environmental relevance of the
compounds (e.g. ecotoxicity). For this reason we have
started to synthesize some of the natural products. Re-
cently, we have prepared the natural heptachloro com-
pound Q1 (Wu et al., 2002), and in this study we present
the synthesis of 4,6-dibromo-2-(20,40-dibromo)phenoxy-
anisole (BC-2) (Fig. 2). BC-2 and six further brominated
phenoxyanisoles have been previously synthesized by
Francesconi and Ghisalberti (1985) in a four step reac-
tion as intermediate products on the way to achieve
brominated phenoxyphenols. To simplify the method we
have applied the iodonium reaction suggested by Marsh
et al. (1999) for the preparation of individual bromi-
nated diphenyl ethers (BDEs). The reaction partner
2,4-dibromo-6-hydroxyanisole was prepared from 2,4,6-
tribromoanisole (Fig. 2). The crude product was frac-
tionated on 60 g silica (elution with n-hexane) which
yielded neat BC-2. The NMR spectrum of the purified
oil and the chromatographic and mass spectrometric
properties were identical with previous BC-2 isolates
(Francesconi and Ghisalberti, 1985; Cameron et al.,
2000).
The determination of non-polar brominated com-
pounds in environmental samples––irrespective as to
whether they are natural or anthropogenic in origin––is
different to chlorinated compounds. Sample preparation
(extraction and elimination of sample matrix) of both
classes can be performed in the same way, but chlori-
nated and brominated compounds behave differently
on silica. Activated silica (or deactivated with small
amounts of water) is often used for the separation of
aromatic organochlorines (PCBs) from alicyclic chlo-
ropesticides (DDT, chlordane, HCH, toxaphene). Usu-
ally, chlorinated aromatic compounds are targeted with
n-hexane in the first fraction, and the brominated ana-
logues have to be eluted with a more polar solvent. In
that way they are found in a fraction which also contains
the chloropesticides. It seems, however, that this is dif-
ferent in NP-HPLC where the brominated and chlori-
nated aromatics elute together. The combination of both
techniques can thus be used for the selective enrichment
of brominated aromatics. This can be necessary since the
concentrations of brominated compounds in marine
samples are often lower than those of the chlorinated
compounds. Therefore, Br-selective GC/MS methods
are required while many chlorinated compounds can be
determined with GC/ECD. The most sensitive technique
for detection of non-polar brominated compounds is
GC/ECNI-MS (Crow et al., 1981; Buser, 1986). Chlo-
rinated and brominated compounds alike form abun-
dant fragment ions in the low mass range. However,
intense fragment ions at high mass are often missing in
the spectra of organobromines, so that the bromide ion
GC/MS analysis of later fractions of the silica
fractionation resulted in the detection of a second
tetrabromophenoxyanisole (RRIBC-2 on DB-5 column ¼
0.9485) and
a
tribromophenoxyanisole (RRIBC-2
¼
0.8153). The tetrabromo isomer of BC-2 may be ex-
plained by formation of the p-hydroxyanisole instead
of the o-hydroxyanisole shown in Fig. 2. Coupling
of this dibromo-p-hydroxyanisole with the 2,20,40,40-
tetrabromodiphenyl iodonium chloride would result in
2,6-dibromo-4-(20,40-dibromo)phenoxyanisole. The by-
product which eluted significantly earlier from DB-5 like
columns (see RRI, above) has not been identified in
environmental samples which would be plausible in the
case of the structural suggestion given above since all
natural brominated anisoles identified to date possess
the anisole group adjacent to the phenoxy group. The EI
mass spectra of BC-2 and its isomer (Fig. 3a) were al-
most identical but the ECNI-MS showed one remark-
able difference. The molecular ion at m=z 512 was not
€
is often used for quantification of BFRs (Sellstrom,
1999; de Boer et al., 2000). This method also allows
detection of natural organobromines (Vetter, 2001).
A major drawback in the analysis of natural halo-
genated compounds is that most of them are not avail-
able as reference standards. This also impedes the
OMe
OMe
Br
Br
Br
OH
Br
Br
1
Br
OMe
Br
Br
Br
O
3
Cl-
Br
Br
Br
+
2
I
BC-2
Br
Br
Br
Fig. 2. Scheme of the synthesis of BC-2.

W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
427
Irel
B1097W.D: scan 1871
(a)
[M-2Br]+ [M-Br-CH3]+
[M]+
[M-2Br]2+
354
418
512
311
100
150
m/z -> 50 100 150 200 250 300 350 400 450 500
Irel
(b)
B1097W.D: scan 1542
340
233
170
276
434
m/z-> 50
100
150
200
250
300
350
400
79/81
A3661W.D: Scan 273
Irel
(c)
161
266
264
355
350
435/437
400 450
m/z-->
100
150
200
250
300
500
Fig. 3. GC/MS investigation of two byproducts of the BC-2 synthesis. EI-MS of (a) the tetrabromo isomer and (b) the tribromo
congener as well as (c) ECNI-MS of the tetrabromoisomer.
observed in the ECNI-MS of the byproduct (Fig. 3c)
which is different to BC-2 (Vetter et al., 2001b). The
highest visible fragment ion in the ECNI-MS of the
byproduct was found at m=z 433 (i.e. [M–Br]À). Inter-
estingly, such an ECNI-MS fragmentation pattern has
been reported previously for BC-3 which is not identical
with the byproduct and whose structure is still unknown
(Vetter, 2001). However, the similarity of the ECNI
mass spectra of BC-3 and the BC-2 byproduct supports
the earlier prediction that BC-3 is a tetrabromophen-
oxyanisole as well (Vetter, 2001). BC-3 and the BC-2
isomer can be distinguished by the ECNI-MS fragment
ion at m=z 264 (formally [C7H6Br2O]À or [M-dibromo-
phenoxy+H]À) which was found for BC-2 and its isomer
but not in the ECNI-MS of BC-3. This may serve as an
indication that BC-3 has a 3:1 distribution of bromine
on the phenyl rings. MeO-BDEs which fulfill this crite-
rion were previously identified by natural products
chemists (Handayani et al., 1997). A further tetra-
bromophenoxyanisole detected in the Baltic Sea (Hagl-
und et al., 1997) was identified as 3,5-dibromo-2-(20,
40-dibromo)phenoxyanisole (Asplund et al., 1999).
BC-2 and BC-11 have been previously identified in
Australian sponges (Cameron et al., 2000). It is curious

428
W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
that sponges, i.e. species largely without any lipids,
should be the producer of highly lipophilic compounds
such as BC-2. There is some indication in the literature
that it is not the sponges but cyanobacteria living on the
sponge (symbiosis) that are responsible for the produc-
tion of brominated secondary metabolites (Unson et al.,
1994). It has been suggested that Oscillatoria may be the
originate producer of BC-2 (Moore et al., 2002). Os-
cillatoria have reasonably high lipid content which
would make this source of production plausible. How-
ever, another explanation is suggested here. We submit
that sponges might be involved in the production of the
brominated phenoxyphenols and that the possible sym-
biont (cyanobacteria) produces the methylether (anisole)
from the phenoxyphenols. This would be a symbiotic
synthesis by sponge and bacteria. However, distin-
guishing the role of the two organisms in the produc-
tion of brominated phenoxyphenols and other natural
products is not simple. For such studies it may be
helpful to note that standards of brominated phen-
oxyphenols can be synthesized from the respective
phenoxyanisoles by treatment with BBr3 in 1,2-dichlo-
roethane (Francesconi and Ghisalberti, 1985). Assuming
that two organisms are involved we would expect
seasonal variations in the ratio of brominated phen-
oxyanisoles and brominated phenoxyphenols. A closer
study of these ratios may thus provide the key to the
understanding of these natural processes. Furthermore,
brominated phenoxyanisoles can be regarded as con-
densation products of brominated phenols and anisoles.
In fact, brominated anisoles (and particularly tribro-
moanisole) have been previously detected in numerous
samples such as fish and mussels from the North Sea
(Rimkus and Wolf, 1991; Rimkus and Wolf, 2001), birds
of prey (Herzke et al., 2001), as well as Antarctic
(Wittlinger and Ballschmiter, 1990; Vetter et al., 2002a)
and Arctic air (Vetter et al., 2002a). The recent identi-
fication of an organism that produces TBA (Flodin and
Whitfield, 1999, 2000) supports the fact that some resi-
dues of TBA are natural although there are also an-
thropogenic sources.
Br
OMe
Br
O
MeO
Br
Br
Br
(b)
OMe
Br
Br
O
HO
Br
Br
OMe
(a)
Br
O
O
Br
Br
(c)
Fig. 4. Possible formation of BC-11 (b) from the tetrabromo-
methoxyphenoxyphenol (a) and reaction of this to the bromi-
nated methoxy dibenzo-p-dioxin (c) which has the same
molecular mass as BC-1.
these two interpretations we have applied chromato-
graphy on charcoal (see Section 2). Using this method, it
was shown that BC-1 eluted together with BC-2 and BC-
3 into the n-hexane fraction. If BC-1 would have been
the tetrabromomethoxydibenzo-p-dioxin (Fig. 4c), this
planar molecule would have been retained on the col-
umn. Chromatography of planar molecules from char-
coal is displacement, so that planar molecules such as
dioxins will only be eluted with P-electron containing
solvents such as toluene (but not n-hexane). Another
possibility to distinguish between the two compounds is
high resolution mass spectrometry. The mass difference
of 0.036 u (exact masses of C13H6Br4O3 and C14H10-
Br4O2 are 525.705087 and 525.741472, respectively) can
be distinguished with this technique.
The identity of further BCs detected in Australian
samples was also partly resolved. BC-10 was identified
as the Br4,Cl2-dimethyl-2,20-bipyrrole discovered by
Tittlemier et al. (1999) and which was recently synthe-
sized by Gribble et al. (1999). Along with these com-
pounds in Australian samples which have also been
detected in samples from other continents, further po-
tential natural products are the unknown tetrabromo
compound UBC-1 detected in samples from the Arc-
tic (Vetter, 2001). Another group of bioaccumulative
natural products are mixed halogenated compounds.
Recently, we have described a dibromotrichloro
monoterpene (MHC-1) in samples from different loca-
tions in the world (Vetter et al., 2001a). Two compounds
with the molecular formula of MHC-1 (C10H13Br2Cl3)
were previously isolated from the red algae Plocamium
cartilagineum (Stierle and Sims, 1979; Higgs et al., 1997).
It was not possible to verify that MHC-1 is identical
with one of the two monoterpenes produced by red algae
Assuming that a two step reaction, i.e. (a) formation
of phenols and (b) formation of anisoles is occurring, the
precursor of BC-11 may be the brominated methoxy-
phenoxyphenol shown in Fig. 4a. This tetrabromo-
methoxyphenoxyphenol may then be transformed into
3,5-dibromo-2-(30,50-dibromo,20-methoxy)phenoxyani-
sole (BC-11) (Fig. 4b). On the other hand, the tet-
rabromomethoxyphenoxyphenol may also result in a
brominated methoxy dibenzo-p-dioxin (Fig. 4c). The
molecular weight (m=z 526) of the brominated methoxy
dibenzo-p-dioxin (C13H6Br4O3) is the same as that pre-
viously determined for BC-1 (Vetter et al., 2001b). Ini-
tially, BC-1 has been described as
a brominated
phenoxyanisole with an additional ‘‘CH2’’ unit (C14H10-
Br4O2) (Vetter et al., 2001b). To distinguish between

W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
429
similar to the aliphatic monoterpene (Fig. 5b) were also
reported in the literature (Gribble, 1998). A dibromo-
trichloro monoterpene isolated from Portieria horn-
emannii has a very pronounced anticancer activity
against human cancer cell lines (Fuller et al., 1992;
Gribble, 1998). In view of these reports, it seems to be
likely that MHC-1 possesses similar biological activity.
However, a disadvantage of the mixed halogenated
monoterpene products in general and particularly of the
compounds with aliphatic backbone is that their syn-
thesis is difficult. Therefore, toxic evaluation of MHC-1
and further mixed halogenated compounds detected in
samples (Vetter, 2001; Vetter et al., 2002a) (see also
below) without identification of the natural producer
and isolation of the compounds from natural organisms,
is not possible at this time.
Cl
Cl
(a)
(c)
(b)
(d)
Cl
Cl
Cl
Br
Br
Br
Cl
Br
Cl
Me
Cl
Cl
Me
Br
Cl
Cl
H
Cl
Fig. 5. Structures of MHC-1 related compounds. (a,b) Natural
products of P. cartilagineum isomeric to MHC-1, (c) telfairine
and (d) procomene.
(Fig. 5a,b). However, our identification of MHC-1 in the
Antarctic, Europe, Northern Europe, Greenland Sea
and other places agrees with the regions where P. car-
tilagineum is sedentary.
The brominated natural products have properties
similar to brominated diphenyl ethers. Therefore, it is an
important task to elucidate if the natural and the an-
thropogenic brominated aromatics may co-elute. Fig. 6
shows the GC/ECNI-MS chromatogram of a sample
extract of human milk from a Faeroese woman who
frequently consumed whale blubber and meat as well as
fish (Abraham et al., 1995). Interestingly, this samples
contained a variety of new and known brominated
compounds. At short retention time a dibromochloro-
anisole was identified on the basis of the full scan mass
spectrum. Note that the retention time was very similar
to TBA, which was not detected in the sample. Next
to MHC-1, another mixed halogenated compound
(MHC-X) was identified on the basis of the [BrCl]À
fragment ion (Vetter, 2001). At higher retention times,
several polybrominated diphenyl ethers were determined
(BDE-47, 99, 100, and 154). The most abundant signal
originated from the natural product BC-3. The analysis
of this sample also demonstrates the interference of
BDEs with halogenated natural products on the most
It is noteworthy, that one of the two structures are
very similar with telfairine (Fig. 5c) and procomeme
(Fig. 5d), two known monoterpenes which have been
previously isolated and whose chemical properties have
been studied (Stierle and Sims, 1979; Higgs et al., 1997).
It was found that telfairine is 100% lethal to mosquito
larvae at 10 ppm, and that procomene is a fourfold
stronger insecticide than the known anthropogenic pes-
ticide lindane (Gribble, 1998). The monoterpene shown
in Fig. 5b and four further structurally similar mono-
terpenes from P. cartilagineum were tested with the
Ames test for mutagenicity since structurally similar
anthropogenic substances such as 1,2-dibromo-3-chloro-
propane (DBCP) induced stomach cancer in test rats
and mice (Leary et al., 1979). The compound shown in
Fig. 5b was mutagenic in the Ames test as were the other
halogenated monoterpenes. Using the S9-mix, the com-
pound depicted in Fig. 5b was active with strain TA100
but not TA98. Further compounds with structures
A3490W.D: m/z 79
BC-3
Dibromo-
chloro-
BDE 47BC-10
BC-11/
BDE 99
anisole
“79“/
BDE BDE
100 154
BC-2
BC-1
MHC-X MHC-1
20.00
10.00
30.00
40.00 [min]
Fig. 6. GC/ECNI-MS SIM chromatogram (m=z 79) showing natural and xenobiotic brominated compounds detected in a sample of
human milk from the Faeroe Islands.

430
W. Vetter, W. Jun / Chemosphere 52 (2003) 423–431
frequently used GC stationary phase for the deter-
mination of organohalogen compounds. BDE–99
co-eluted with BC-11 and BDE-154 co-eluted with an
unknown brominated that did not form low-mass frag-
ment ions other than [Br]À (‘‘type 79’’, Vetter, 2001).
Such coelutions may cause false-positive identification
of BFRs in wildlife.
regulation in population dynamics may be obtained.
This distinguishes them from the halogenated xenobio-
tica which can be produced at any amount and con-
centration. However, the mode of bioaccumulation
seems to be the same for anthropogenic and natural
organohalogens. Finally, we need to state that there is a
lack of research on the relevance of the bioaccumulation
of naturally produced organohalogens.
Our previous work was mainly restricted to top
predators of marine food webs. We have also started
screening of samples from lower tropic levels (Fig. 7).
The cod sample from the Baltic Sea showed an intense
signal for TBA. Next to the natural chlorinated com-
pound Q1 (identified by Q1-selective m=z 386), we also
identified BDE-47 and several other compounds (Fig.
7a). The mussel sample from Mexico also showed TBA
and Q1 as well as a number of unknown brominated
compounds some of which may be tribromo compounds
(Fig. 7b).
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