Metabolites from the biodegradation of 1, 6-hexanediol dibenzoate, a potential green plasticizer, by Rhodococcus rhodochrous

Article abstract of DOI:10.1002/jms.1541

Metabolites from the biodegradation of a potential plasticizer 1, 6-hexanediol dibenzoate in the presence of n-hexadecane as a co-substrate by the common soil organism Rhodococcus rhodochrous were identified using GC/MS and Fourier transform mass spectros

Full text of DOI:10.1002/jms.1541

Research Article
Received: 14 November 2008
Accepted: 16 November 2008
Published online in Wiley Interscience: 5 January 2009
(
www.interscience.com) DOI 10.1002/jms.1541
Metabolites from the biodegradation
of 1,6-hexanediol dibenzoate, a potential
green plasticizer, by Rhodococcus rhodochrous
a
b∗
a
Azadeh Kermanshahi Pour, Orval A. Mamer, David G. Cooper,
a
c
Milan Maric and Jim A. Nicell
Metabolites from the biodegradation of a potential plasticizer 1,6-hexanediol dibenzoate in the presence of n-hexadecane as a
co-substrate by the common soil organism Rhodococcus rhodochrous were identified using GC/MS and Fourier transform mass
spectroscopy (FTMS) techniques. Trimethylsilylation of compounds from the biodegradation broth permitted detection of the
following metabolites: 1-hexadecyl benzoate, 6-benzoyloxyhexanoic acid, 4-benzoyloxybutanoic acid, 6-benzoyloxyhexan-1-
ol and benzoic acid. The presence of these metabolites was confirmed by repeating the biodegradation with 1,6-hexanediol
2
di[ H5]benzoate, by measurement of their exact masses in FTMS and by comparison with available authentic materials. The
results show that biodegradation of 1,6-hexanediol dibenzoate by R. rhodochrous does not lead to the accumulation of
persistent metabolites as has been reported for commercial dibenzoate plasticizers. Copyright ꢀc 2009 John Wiley & Sons, Ltd.
Keywords: 1,6 hexanediol dibenzoate; plasticizer; biodegradation; metabolites; GC/MS; Fourier transform mass spectroscopy
Introduction
In recent years, dibenzoate plasticizers such as diethylene
glycol dibenzoate (D(EG)DB) and dipropylene glycol dibenzoate
Theinteractionofmicroorganismswithxenobioticchemicalsinthe
environment is a critical issue that must be studied when assessing
their toxic impacts on ecological systems and human health.[1–4]
Incomplete biodegradation of the parent compounds in the
environment may lead to their accumulation and the production
of metabolites of increased mobility, toxicity and persistence.[5–8]
Therefore, in order to fully assess the impacts of these xenobiotics
in the environment, it is important to identify the full range of
compounds that are produced through biodegradation and to
assess their toxicity and biodegradability.
(
D(PG)DB) have been proposed as alternatives to the more
commonly used compounds because they tend to degrade
more rapidly under the action of common microorganisms.
[19,20]
Although this tendency appears to make them attractive as
alternatives to phthalates and adipates, it has been shown that the
incomplete microbial hydrolysis of D(EG)DB) and D(PG)DB when
microorganisms are growing on glucose as a primary co-substrate
leads to the accumulation of diethylene glycol monobenzoate
anddipropyleneglycolmonobenzoate,respectively,whichexhibit
[21]
significant toxicity.
In addition, the development of alternative compounds to
replace conventional plasticizers requires monitoring of the con-
sequencesoftheirbiodegradationtoensurethattheirmetabolites
have minimal toxicological impacts in the environment. Plas-
ticizers, which are the most widely used additives in polymer
manufacturing,[9] have raised serious health and environmental
However, therapiddegradationofthedibenzoatesindicatethat
a potential route to the development of a ‘green’ plasticizer may
be to start with the basic structure of the more easily degraded
dibenzoate plasticizer and modify it to reduce the accumulation
of metabolites when undergoing biodegradation. Therefore, the
aim of this study was to monitor the biotransformation of
[
10,11]
concerns in recent years.
In addition, the concerns raised
1,6-hexanediol dibenzoate, a potential plasticizer, by Rhodococcus
above about the potential impacts of metabolites have been
shown to be the case for a number of widely used commercial
plasticizers including phthalates and adipates.[4,6–8] Studies of
the biodegradation of phthalate and adipate plasticizers have re-
portedtheproductionofseveraldifferentmetaboliteswithgreater
rhodochrous, a common soil organism, in the presence of
hexadecane as a primary carbon source and to identify all of
the metabolites created during biodegradation. Low-resolution
[
4,6–8]
toxicity than the parent compounds.
For example, 2-ethylhexanoic acid, a potent peroxisome
∗
Correspondence to: Orval A. Mamer, Mass Spectrometry Facility, McGill
University, 740 Dr Penfield Avenue, Montreal, QC, Canada H3A 1A4.
E-mail: orval.mamer@mcgill.ca
proliferator,[
12,13]
was identified from the biodegradation of di-2-
ethylhexyl adipate, di-2-ethylhexyl phthalate and di-2-ethylhexyl
terephthalate.[ Mono-2-ethylhexyl phthalate, a metabolite ex-
a Department of Chemical Engineering, McGill University, 3610 University Street,
Montreal, QC, Canada H3A 2B2
6]
[
14]
pected in the biodegradation of di-2-ethylhexyl phthalate,
is
[
15]
b MassSpectrometryFacility,McGillUniversity,740DrPenfieldAvenue,Montreal,
QC, Canada H3A 1A4
classified as an endocrine disruptor.
Detection of these and
related metabolites of phthalates and adipates in mice, rats and
human plasma and urine has led to greater concerns and stricter
c Department of Civil Engineering and Applied Mechanics, McGill University, 817
Sherbrooke Street West, Montreal, QC, Canada H3A 2K6
[
16–18]
environmental regulations.
J. Mass. Spectrom. 2009, 44, 662–671
Copyright ꢀc 2009 John Wiley & Sons, Ltd.

Metabolites of 1,6-hexanediol dibenzoate
Figure 1. NMR spectrum of synthesized 1,6-hexanediol dibenzoate. Relative integration of protons on indicated carbon atom (A : B : C : D : E : F =
2
: 2 : 1 : 2 : 2 : 2). A, B, C exhibit the expected patterns for a phenyl group. D and F are triplets and E is a multiplet (assumed to be a triplet of triplets) as
expected for this part of the structure.
Figure 2. Gas chromatograms obtained for the underivatized broth extract (panel A) and for the extract after trimethylsilylation (panel B). In the former,
peaks are identified as 1: hexadecane; 2: 1-hexadecyl benzoate; 3: 1,6-hexanediol dibenzoate. In the latter, peaks are identified as 1: benzoic acid TMS
derivative; 2: 1,6-hexanediol TMS derivative; 3: pentadecane added to the extract as a retention time marker; 4: hexadecane added to the broth as a
co-metabolite; 5: 4-benzoyloxybutyric acid TMS derivative; 6: 6-benzoyloxyhexan-1-ol TMS derivative; 7: 6-benzoyloxyhexanoic acid TMS derivative; 8:
1
1
,6-hexanediol dibenzoate and 9: 1-hexadecyl benzoate. Other eluting peaks in B originate in the solvent and derivatizing reagent. The elution order for
-hexadecylbenzoate and 16-hexanediol dibenzoate is reversed by switching between DB-1 and HP-5 columns.
J. Mass. Spectrom. 2009, 44, 662–671
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A Kermanshahi Pour et al.
Figure 3. Electron ionization mass spectra of (A) 1,6-hexanediol diben-
2
zoate and (B) 1,6-hexanediol di[ H5]benzoate isolated from the incubation
mixtures. Their mass spectra and GC retention times are identical to those
for the diesters synthesized for this study. The intensities of ions above m/z
220 are multiplied by 20.
GC/MS with electron ionization was used for the identification of
the metabolites as their trimethylsilyl (TMS) derivatives. Fourier
transform mass spectroscopy (FTMS) was also used to obtain their
underivatized accurate masses with electrospray ionization (ESI).
Experimental
Chemicals and reagents
Figure 4. Electron ionization mass spectra of (A) 1-hexadecyl benzoate,
2
(
B) synthesized 1-hexadecyl benozate and (C) 1-hexadecyl [ H5]benzoate.
1
,6-Hexanediol 99%, 1-hexadecanol 99%, n-hexadecane 99%
The intensities of ions above m/z 240 are multiplied by 50.
and benzoyl chloride 99% were purchased from Sigma-
2
Aldrich (Oakville, ON, Canada). [ H5]Benzoyl chloride 99.1
atom% D was purchased from CDN isotopes (Montreal, QC,
Canada). Bacto Brain/Heart infusion and yeast extract were
obtained from Difco Microbiology (Montreal, QC, Canada)
and Fisher Scientific (Montreal, QC, Canada), respectively.
Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was purchased from
Chromatographic Specialties (Brockville, ON, Canada). Pentade-
canewaspurchasedfromA&CAmericanChemicals(Montreal, QC,
Canada). All other chemicals were obtained from Fisher Scientific.
Synthesis of 1,6-hexanediol dibenzoate and 1,6-hexanediol
di[2H5]benzoate
1,6-Hexanediol dibenzoate was synthesized by refluxing 5 g of
1,6-hexanediol with 20 ml of benzoyl chloride (4 equivalents)
under nitrogen in 120 ml of acetone in a round bottom flask for
7 h. The reaction mixture was cooled to room temperature and
then diluted with 100 ml of chloroform. The mixture was washed
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Metabolites of 1,6-hexanediol dibenzoate
Figure 5. Electron ionization mass spectra of the TMS derivatives of
Figure 6. Electron ionization mass spectra of the TMS derivatives of (A) 6-
benzoyloxyhexan-1-ol and (B) 6-[ H ]benzoyloxyhexan-1-ol isolated from
2
2
5
(
A) 6-benzoyloxy-hexanoic acid and (B) 6-[ H5]benzoyloxyhexanoic acid
isolated from the incubation mixtures. The intensities of ions above m/z
10 are multiplied by 20.
the incubation mixtures.
2
mixture was cooled to room temperature and then diluted with
threetimeswith100 mlofasaturatedsodiumbicarbonatesolution
and concentrated to a yellow oil, which, on standing, yielded an
off-white powder. This was re-crystallized from heptane.
1
1
00 ml of chloroform. The mixture was washed three times with
00 ml of a saturated sodium bicarbonate solution. The solvent
was then evaporated and 1-hexadecyl benzoate was crystallized
from the residue. The proton NMR spectrum obtained from the
synthesized 1-hexadecyl benzoate was in agreement with the
spectrum expected for this compound.
2
The synthesis of 1,6-hexanediol di[ H5]benzoate was achieved
in a similar manner by reacting 1,6-hexanediol (0.39 g) with 1.2 ml
2
of benzoyl-[ H5]-chloride (3 equivalents) in 11 ml of acetone. The
procedure used for work-up and re-crystallization is described
above.
The proton nuclear magnetic resonance (NMR) spectra of
the synthesized 1,6-hexanediol dibenzoate and 1,6-hexanediol
di[ H5]benzoate were consistent with the spectra expected for
these compounds (Fig. 1). The EI spectrum of the unlabelled
diester was virtually identical to the spectrum published in the
NIST/EPA/NIH 1998 Mass Spectral Library of the United States
Department of Commerce.
Biodegradation studies
2
Biodegradation of 1,6-hexanediol dibenzoate or 1,6-hexanediol
2
di[ H5]benzoate was conducted in 500 ml Erlenmeyer flasks with
a sponge cap. The medium for the experiments consisted of
100 ml of the sterilized minimum mineral salt medium (MMSM)
and 0.1 g/l of yeast extract and 2.5 g/l n-hexadecane. Either
2
1,6-hexanediol dibenzoate or 1,6-hexanediol di[ H5]benzoate
(
3 mmol/l)wereaddedtotheflasksindividuallybeforeautoclaving.
Synthesis of 1-hexadecyl benzoate
The MMSM contained 4 g/l NH4NO3, 4 g/l KH2PO4, 6 g/l Na2HPO4,
1
1
-Hexadecyl benzoate was synthesized by reacting 8 g of
-hexadecanol with 5 ml of benzoyl chloride (1.3 equivalents)
0.2 g/lMgSO ·7H2O, 0.01 g/lCaCl2·2H2O, 0.01 g/lFeSO4·7H2Oand
4
0.014 g/l disodium ethylenediamine-tetraacetic acid.
in 120 ml of refluxing acetone in a round bottom flask for
h. The reaction was carried out under nitrogen. The reaction
Rhodococcus rhodochrous ATCC 13 808 was obtained from the
American Type Culture Collection (ATCC, Rockville, MD, USA) and
7
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A Kermanshahi Pour et al.
◦
was stored at −70 C in plastic vials containing 20% glycerol and
added to enhance cationization and to provide a secondary mass
scale calibrating ion (Na2I , m/z 172.8835). The ‘Z’-spray source
+
a sterile growth medium of Bacto Brain/Heart infusion broth.
To prepare the initial inoculum, the contents of a vial were
thawed and transferred to a 500-ml shaker flask containing
sterile growth medium composed of Brain/Heart infusion (30 g/l
Brain/Heart infusion broth in 100 ml of distilled water) and then
incubated on a rotary shaker (Series 25, New Brunswick Scientific,
used capillary and cone voltages of 3899 and 30 V, respectively.
Ions were accumulated in the hexapole for 300–1500 ms with a
rod voltage of 70 V. For the transfer of ions to the ICR cell through
the hexapole ion guide, the low mass range coil with a frequency
of 3020 kHz was used along with a voltage of 80 V. For detection,
ions were excited through an arbitrary waveform in a range of m/z
100–1000 with an amplitude of 135 V(b-p); the analog to digital
conversion (ADC) rate for the MS was 2 MHz for a scan range of
m/z 75–500. Transients were 1M data points long. A waiting time
of 5 s before the detection step was used to allow the pressure in
the ICR cell to return to its nominal value of 2 × 10− torr.
◦
Edison, NJ, USA) set at 250 rpm and 30 C. After a day, a new
shaker flask containing 100 ml of 30 g/l sterile growth medium of
Brain/Heart infusion in distilled water was inoculated with 1 ml of
the initial inoculum.
When exponential growth was reached, this microbial culture
was used to inoculate 100 ml of the sterilized MMSM containing
9
0
.1 g/l yeast extract and 2.5 g/l n-hexadecane. This was used to
inoculate the shaker flasks containing either 1,6-hexanediol diben-
zoate or 1,6-hexanediol di[ H5]benzoate for the biodegradation
GC/FID analyses
2
study. The shaker flasks were incubated for a period of 7 days on a
rotary incubator shaker set at 250 rpm and 30 C.
Aliquots (1 µl) of the chloroform extracts were analyzed in a Varian
CP-3800 gas chromatograph equipped with a 30 m × 0.32 mm
i.d. fused silica 8CB column (Varian, Montreal, QC, Canada)
◦
◦
◦
programmed to 300 C at 10 C after a 2-min hold initially at
Sample preparation for GC/MS and GC/FID analyses
◦
◦
◦
4
0 C. The injection port and FID were kept at 250 C and 300 C,
Over the course of biodegradation of 1,6-hexanediol dibenzoate,
triplicatesamplesof3 mleachweretakenfromthebiodegradation
broth every day. The samples were adjusted to pH 2 through the
addition of sulfuric acid and extracted with 3 ml of chloroform.
For GC/MS analysis, the extracts were evaporated to dryness
under a dry nitrogen stream and the residues were taken up in
respectively. Helium was used as a carrier gas at a flow rate of
1.5 ml/min. The concentration of the metabolites were estimated
by GC/FID.
Results and Discussion
5
0 µl of anhydrous pyridine. Trimethylsilyl (TMS) derivatives were
preparedbytheadditionof50 µlofBSTFAtothepyridinesolutions
in capped auto-injector vials, which were heated in an aluminum
block at 60 C for 15 min. For GC/ flame ionization detection (FID)
1,6-Hexanediol dibenzoate was synthesized in an attempt to
develop a more environmentally benign version of the standard
dibenzoate plasticizers. It has been shown that analogous com-
pounds can be converted to stable toxic metabolites by microor-
◦
analysis, chloroform extracts of the samples were used without
derivatization.
[
21]
ganisms while growing on an easily metabolized substrate. The
purpose of this work was to determine whether co-metabolism
of 1,6-hexanediol dibenzoate by a typical soil microorganism R.
rhodochrous resulted in the production of stable metabolites.
Figure 2 shows the total ion current GC of an extract
of an experiment using 1,6-hexanediol dibenzoate without
derivatization (panel A) and after trimethylsilylation (panel B).
Retention times and elution order are different in panel A and
GC/MS analyses
Aliquots (1 µl) of the underivatized extracts were analyzed in low-
resolution GC/MS mode with a GCT (Micromass, Manchester, UK)
fitted with a 30-m HP-5 capillary column having a 0.32-mm i.d.
and 0.25-µm film thickness. The temperature was programmed
◦
◦
◦
◦
from 80 C after 1-min hold to 300 C at 10 C/min followed by
panel B due to the use of a HP-5 column and 10 C program (panel
◦
◦
a bake-out period of 6 min at 300 C. The injector was operated
in 1 : 100 split mode at 250 C with a constant helium pressure
of 70 kPa. The GC re-entrant temperature was 250 C. The EI ion
source was operated at 70 eV and 200 C.
A) versus a DB-1 column and a 5 C temperature program for the
◦
derivatized sample (panel B).
◦
Figure 3A and B shows the EI mass spectra of 1,6-hexanediol
◦
2
dibenzoate and 1,6-hexanediol di[ H5]benzoate, respectively.
TMS-derivatized extracts and the synthesized 1-hexadecyl
benzoate were analyzed in GC/MS mode on a 30-m, 0.25-mm
i.d. DB-1 column operated as described above. The scan range
was m/z 80–600 to avoid the intense but uninformative m/z 73
common to TMS derivatives.
Weak molecular cations at m/z 326 and 336 were observed in their
mass spectra, respectively. Fragmentation pathways are proposed
in Scheme 1. Fragment ions at m/z 204/209 are formed by loss
of benzoic acid via a McLafferty mechanism and with a possible
subsequent elimination of the elements of formaldehyde to yield
ions at m/z 174 and 179. Formaldehyde elimination, as proposed
in Scheme 1, is not observed in the dibenzoates of 1,5-pentanediol
or 1,4-butanediol (data not shown, available in the NIST/EPA/NIH
1998 Mass Spectral Library), and this may be related to the alkyl
chain length. There are three possible precursor ions to m/z
FTMS analyses
High-resolution measurements of the underivatized extracts were
made in positive ion electrospray mode with an IonSpec 7.0 tesla
FTMS (Lake Forest, CA, USA) calibrated with polyethylene glycol
+
•
174/179; these are m/z 221/226, 204/209 and the M . The first
would violate the even electron ‘rule’, and it is not possible to
decide between the last two on the basis of the present data.
Fragments at m/z 123/128, nominally protonated benzoic acid,
may be formed in a four-centred elimination of a radical olefin.
3
00. The instrument was equipped with a ‘Z’-spray source from
Waters Corporation (Milford, MA, USA), an accumulation hexapole,
a collision cell, a hexapole ion guide, a standard cylindrical ion
cyclotron resonance (ICR) cell and Omega 9 software. The analyses
usedadirectinfusionflowrateof2–3 µl/mininsolutionwith90 : 10
v/v methanol : water. Formic acid (1%) and sodium iodide were
+
Alternatively, m/z 123/128 may have the Ph-C(OH)2 structure.
Subsequent loss of water by m/z 123/128 results in ions at m/z
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Metabolites of 1,6-hexanediol dibenzoate
2
Scheme 1. Proposed fragmentation scheme for 1,6-hexanediol dibenzoate and 1,6-hexanediol di[ H5]benzoate. The second m/z value refers to the
labelled diester.
2
Scheme 2. Proposed mass spectrometric fragmentation scheme for 1-hexadecyl benzoate and 1-hexadecyl [ H5]benzoate. The second m/z value refers
to the labelled ester.
2
1
05/110. Formation of this last ion by a direct C(O)–O fission in
6-[ H5]benzoyloxyhexanoic acid, respectively. In support of this
assignment, Scheme 3 accounts for the major ion fragments and
deuterium labelling found in Fig. 5A and B. Loss of methyl rad-
ical by the molecular radical cations yields m/z 293 and 298.
Subsequent loss of CO2 by m/z 293/298 yields m/z 249/254. M/z
179/184 is formed by the loss of the elements of ε-caprolactone
by m/z 293/298, and goes on to lose CO2 to form m/z 135/140
and Si(CH3)2O resulting in m/z 105/110. M/z 105/110 formation
directly from the molecular cation can neither be excluded here
nor in the case of Scheme 4. M/z 117 is an ion characteristic of
TMS derivatives of compounds with carboxy groups, which cor-
the molecular ion cannot be discounted.
Figure 4AandCisthemassspectraof1-hexadecylbenzoateand
2
1
-hexadecyl [ H5]benzoate, respectively, isolated from the broth.
The mass spectrum of the synthesized 1-hexadecyl benzoate
is shown in Fig. 3B. The proposed fragmentation scheme for
2
1
-hexadecylbenzoateand1-hexadecyl[ H5]benzoateinScheme 2
accounts for the formation of fragments at m/z 123/128 and
1
1
05/110 in a manner parallel to that suggested in Scheme 1 for
,6-hexanediol dibenzoate and 1,6-hexanediol di[ H5]benzoate.
2
Again, as in Scheme 1, although m/z 123/128 is drawn as
protonated benzoic acid, the ion structure may be that of a
α,α-dihydroxybenzyl cation. m/z 105/110 may be formed by H2O
+
[22]
responds to COOTMS.
Weak m/z 117 ions were observed in
both Fig. 5A and B.
+
•
loss or directly from the M . A McLafferty rearrangement in the
molecular cations generates m/z 224/224, which undergo the
sequential loss of two ethylene groups.
Figure 6A and B represents the spectra of the TMS derivatives of
2
6-benzoyloxyhexan-1-ol and 6-[ H5]benzoyloxyhexan-1-ol, which
were expected metabolites in the biodegradation of 1,6-
2
Figure 5A and B shows the mass spectra of the trimethylsily-
lated metabolites identified as 6-benzoyloxyhexanoic acid and
hexanediol dibenzoate and 1,6-hexanediol di[ H5]benzoate, re-
spectively, by analogy to the metabolites reported for related
J. Mass. Spectrom. 2009, 44, 662–671
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A Kermanshahi Pour et al.
2
Scheme 3. Proposed mass spectrometric fragmentation scheme for the TMS derivatives of 6-benzoyloxyhexanoic and 6-[ H5]benzoyloxyhexanoic acids.
The second m/z value refers to the labelled TMS ester.
Scheme 4. Proposed mass spectrometric fragmentation scheme for the TMS derivatives of 6-benzoyloxyhexan-1-ol. The second m/z value refers to the
labelled TMS ester.
commercial plasticizers.[21] Scheme 4 proposes fragmentations
that account for the major ions in the mass spectra. An inter-
esting Me2Si migration appears to occur in the loss of CO2 for
the transition m/z 179/184–135/140. The latter ion is drawn as a
silicon analogue of an α,α-dimethylbenzyl cation, but the actual
structure is not known. M/z 135 is also an intense fragment in the
spectrum of the TMS ester of benzoic acid (NIST/EPA/NIH 1998
Mass Spectral Library) and is similarly formed by loss of CO2 from
m/z 179 produced by methyl radical loss from the molecular cation
radical cations (m/z 280 and 285, not detected), and go on to
lose CO forming m/z 221 and 226. Ions at m/z 179/184, 135/140
2
and 105/110 likely have the same structures as those proposed
in Scheme 3. M/z 117 was also observed in both Fig. 7A and B
indicating the presence of a carboxyl group.
Figure 8A and B is the mass spectra of the TMS derivatives
2
of benzoic and [ H5]benzoic acids, respectively, found in the
derivatized extracts. The molecular ions at m/z 194 and 199 show
fragmentations similar to those found in Figs 5, 6 and 7.
(m/z 194), both metastable confirmed (data not shown).
The experimental and calculated exact masses obtained for
ThespectraoftheTMSderivativesof4-benzoyloxybutanoicacid
2
2
1,6-hexanediol dibenzoate and 1,6-hexanediol di[ H ]benzoate
and 4-[ H5] benzoyloxybutanoic acids are illustrated in Fig. 7A and
B, and show homology with the spectra of 6-benzoyloxyhexanoic
5
and their metabolites are presented in Table 1. The difference
2
and 6-[ H5]benzoyloxyhexanoic acids (Fig. 5A and B). M/z 265 and
between the calculated and experimental masses was 2.3 ppm or
better.
2
70 are formed by the loss of methyl radical by the molecular
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Metabolites of 1,6-hexanediol dibenzoate
Figure 8. Electron ionization mass spectra of the TMS derivatives of
(A) benzoic acid and (B) [ H ] benzoic acid isolated from the incubation
mixtures.
2
5
Figure 7. Electron ionization mass spectra of the TMS derivatives of (A) 4-
benzoyloxybutanoic acid and (B) 4-[ H5] benzoyloxybutanoic acid isolated
from the incubation mixtures.
2
of benzoate hydrolyzed from 1,6-hexanediol dibenzoate or
its metabolites to hexadecanol, a metabolite of hexadecane
degradation. This is consistent with the fact that biodegradation
None of the above metabolites were observed in abiotic control
experiments, eliminating the possibility of the formation of any of
these metabolites by chemical hydrolysis or oxidation.
2
of 1,6-hexanediol di[ H5]benzoate resulted in formation of 1-
Table 2 contains data for the highest observed concentrations
and maximum lifetime for each of the metabolites. All of these
metabolites eventually disappeared. The most important of these
is the monoester, 6-benzoyloxyhexan-1-ol. This compound is
analogous to the monoesters produced by co-metabolism of the
2
hexadecyl [ H5]benzoate.
This ester of 1-hexadecanol is not a potential problem. It
is an artefact arising from the use of hexadecane as the
primary carbon and energy source. Hexadecane was convenient
for these experiments because R. rhodochrous grows well on
[
21]
commercial plasticizers D(EG)DB and D(PG)DB.
However, the
[4]
hydrocarbons and a hydrophobic substrate helps to disperse
monoesters (diethylene glycol monobenzoate and dipropylene
glycol monobenzoate) from biodegradation of the commercial
plasticizers were not only resistant to further biodegradation
but were also shown to exhibit significant toxicity in screening
assays.[ Therefore, the fact that the monoester of 1,6-hexanediol
was only observed in small quantities and also degraded rapidly
supports the hypothesis that this compound may represent a
more environmentally benign dibenzoate plasticizer.
The most long lived of the metabolites in Table 2 and one that
was observed at an order of magnitude greater concentration
than the monoester was 1-hexadecyl benzoate. This is the only
metabolite that cannot originate directly from the degradation of
the water-insoluble plasticizer. However, in an environmental
situation, this benzoate is unlikely to be formed because there will
not be appreciable amounts of alkanes or alcohols present.
21]
Conclusions
GC/MSandFTMSwereusedtoidentifymetabolitesarisingfromthe
biodegradationof1,6-hexanedioldibenzoatebyR.rhodochrous.All
ofthesemetaboliteswereconfirmedbyrepeatingtheexperiments
with deuterium-labelled analogues.
In contrast to commercially available dibenzoate plasticizers,
metabolism of 1,6-hexanediol dibenzoate did not result in
accumulation of persistent metabolites. Furthermore, the most
1,6-hexanediol dibenzoate. It is hypothesized that the formation
of 1-hexadecyl benzoate is the result of an enzymatic conjugation
J. Mass. Spectrom. 2009, 44, 662–671
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
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A Kermanshahi Pour et al.
Table 1. Accurate masses of labelled and unlabelled 1,6-hexanediol dibenzoate and metabolites measured in positive ion electrospray
Compound
Ion Composition
Found
Required
Error (ppm)
1
1
,6-Hexanediol dibenzoate
C20H22O4Na
C20H23O4
349.1410
327.1591
359.2037
337.2222
347.2953
352.3252
259.0942
264.1254
245.1148
250.1457
231.0628
236.0942
123.0441
128.0757
349.1410
327.1591
359.2038
337.2218
347.2945
352.3258
259.0941
264.1255
245.1148
250.1462
231.0628
236.0942
123.0441
128.0754
0.0
0.0
0.3
1.1
2.3
1.7
0.4
0.4
0.0
2.0
0.0
0.0
0.0
2.3
2
2
,6-Hexanediol di[ H10]benzoate
C20H12 H10O4Na
2
C20H13 H10O4
1
1
6
6
6
6
4
4
-Hexadecyl benzoate
C23H39O2
2
2
-Hexadecyl [ H5]benzoate
C23H34 H5O2
-Benzoyloxyhexanoic acid
-[2H5]Benzoyloxyhexanoic acid
-Benzoyloxyhexan-1-ol
-[2H5]Benzoyloxyhexan-1-ol
-Benzoyloxybutanoic acid
-[2H5]Benzoyloxybutanoic acid
C13H16O4Na
2
C13H11 H5O4Na
C13H18O3Na
2
C13H13 H5O3Na
C11H12O4Na
2
C11H7 H5O4Na
Benzoic acid
[2
H5]Benzoic acid
C7H7O2
2
C7H2 H5O2
[
[
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Time of
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-Benzoyloxyhexanoic 0.30 ± 0.04
40
64
87
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acid
[
[
-Benzoyloxybutanoic 0.07 ± 0.01
acid
Benzoic acid
0.20 ± 0.02
40
40
87
1
-Hexadecyl benzoate 0.31 ± 0.01
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a
Initial concentration of 1,6-hexanediol dibenzoate was 3 mmol/l.
Values are the average of triplicate samples. Samples were taken once
b
per day.
[
stable of the metabolites would not be expected to be observed
in the environment. These results support the potential to use
,6-hexanediol dibenozate as a green plasticizer.
[
[
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Acknowledgements
The authors are grateful for the financial support of the Natural
Sciences and Engineering Research Council of Canada and the
EJLB Foundation of Canada. We would also like to thank Mr
Luc Choini e` re, Dr Alain Lesimple and Mr Ranjan Roy for GC/MS
and FTMS analysis. O.M. wishes to acknowledge grants from
the Canadian Institutes for Health Research and the Canadian
Foundation for Innovation. Azadeh Kermanshahi Pour thanks
NSERC, the McGill Engineering Doctoral Award program, and
the Eugenie Ulmer Lamothe fund of the Department of Chemical
Engineering at McGill University for providing scholarships in
support of her Ph.D. studies.
[
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17] H. G. Wahl, Q. Hong, S. Hildenbrand, T. Risler, D. Luft, H. Liebich.
4
-Heptanone is a metabolite of the plasticizer di(2-ethylhexyl)
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